Geometrically restricted 2-indolinone derivatives as modulators of protein kinase activity

ABSTRACT

The present invention relates to novel geometrically restricted 2-indolinones and physiologically acceptable salts thereof which modulate the activity of protein kinases and therefore are expected to be useful in the prevention and treatment of protein kinase related cellular disorders such as cancer.

This application is a division of application Ser. No. 09/385,974 filedAug. 30, 1999 now U.S. Pat. No. 6,525,072.

RELATED APPLICATIONS

This application is related to and claims priority from United StatesProvisional Patent Application serial No. 60/098,660 filed Aug. 31, 1998which is incorporated by reference as if fully set forth herein.

INTRODUCTION

This invention relates generally to organic chemistry, biochemistry,pharmacology and medicine. More particularly, it relates togeometrically restricted 2-indolinone derivatives and theirphysiologically acceptable salts and prodrugs which modulate theactivity of protein kinases (“PKs”) and, therefore, are expected toexhibit a salutary effect against disorders related to abnormal PKactivity.

BACKGROUND OF THE INVENTION

The following is offered as background information only and is notadmitted to be prior art to the present invention.

PKs are enzymes that catalyze the phosphorylation of hydroxy groups ontyrosine, serine and threonine residues of proteins. The consequences ofthis seemingly simple activity are staggering; cell growth,differentiation and proliferation, i.e., virtually all aspects of celllife in one way or another depend on PK activity. Furthermore, abnormalPK activity has been related to a host of disorders, ranging fromrelatively non-life threatening diseases such as psoriasis to extremelyvirulent diseases such as glioblastoma (brain cancer).

The PKs can be conveniently broken down into two classes, the proteintyrosine kinases (PTKS) and the serine-threonine kinases (STKs).

One of the prime aspects of PTK activity is their involvement withgrowth factor receptors. Growth factor receptors are cell-surfaceproteins. When bound by a growth factor ligand, growth factor receptorsare converted to an active form which interacts with proteins on theinner surface of a cell membrane. This leads to phosphorylation ontyrosine residues of the receptor and other proteins and to theformation inside the cell of complexes with a variety of cytoplasmicsignaling molecules that, in turn, effect numerous cellular responsessuch as cell division (proliferation), cell differentiation, cellgrowth, expression of metabolic effects to the extracellularmicroenvironment, etc. For a more complete discussion, see Schlessingerand Ullrich, Neuron, 9:303-391 (1992) which is incorporated byreference, including any drawings, as if fully set forth herein.

Growth factor receptors with PTK activity are known as receptor tyrosinekinases (“RTKs”). They comprise a large family of transmembranereceptors with diverse biological activity. At present, at leastnineteen (19) distinct subfamilies of RTKs have been identified. Anexample of these is the subfamily designated the “HER” RTKs, whichinclude EGFR (epithelial growth factor receptor), HER2, HER3 and HER4.These RTKs consist of an extracellular glycosylated ligand bindingdomain, a transmembrane domain and an intracellular cytoplasmiccatalytic domain that can phosphorylate tyrosine residues on proteins.

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor I receptor (IGF-1R) and insulin receptor related receptor(IRR). IR and IGF-LR interact with insulin, IGF-I and IGF-II to form aheterotetramer of two entirely extracellular glycosylated a subunits andtwo β subunits which cross the cell membrane and which contain thetyrosine kinase domain.

A third RTK subfamily is referred to as the platelet derived growthfactor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR,c-kit and c-fms. These receptors consist of glycosylated extracellulardomains composed of variable numbers of immunoglobin-like loops and anintracellular domain wherein the tyrosine kinase domain is interruptedby unrelated amino acid sequences.

Another group which, because of its similarity to the PDGFR subfamily,is sometimes subsumed into the later group is the fetus liver kinase(“flk”) receptor subfamily. This group is believed to be made up ofkinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1, VEGF-R2),flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1).

A further member of the tyrosine kinase growth factor receptor family isthe fibroblast growth factor (“FGF”) receptor subgroup. This groupconsists of four receptors, FGFR1-4, and seven ligands, FGF1-7. Whilenot yet well defined, it appears that the receptors consist of aglycosylated extracellular domain containing a variable number ofimmunoglobin-like loops and an intracellular domain in which thetyrosine kinase sequence is interrupted by regions of unrelated aminoacid sequences.

Still another member of the tyrosine kinase growth factor receptorfamily is the vascular endothelial growth factor (“VEGF”) receptorsubgroup. VEGF is a dimeric glycoprotein similar to PDGF but hasdifferent biological functions and target cell specificity in vivo. Inparticular, VEGF is presently thought to play an essential role isvasculogenesis and angiogenesis.

A more complete listing of the known RTK subfamilies is described inPlowman et al., DN&P, 7(6):334-339 (1994) which is incorporated byreference, including any drawings, as if fully set forth herein.

In addition to the RTKS, there also exists a family of entirelyintracellular PTKs called “non-receptor tyrosine kinases” or “cellulartyrosine kinases.” This latter designation, abbreviated “CTK,” will beused herein. CTKs do not contain extracellular and transmembranedomains. At present, over 24 CTKS in 11 subfamilies (Src, Frk, Btk, Csk,Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Srcsubfamily appear so far to be the largest group of CTKs and includesSrc, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detaileddiscussion of CTKs, see Bolen, Oncogene, 8:2025-2031 (1993), which isincorporated by reference, including any drawings, as if fully set forthherein.

The serine/threonine kinases, STKs, like the CTKs, are predominantlyintracellular although there are a few receptor kinases of the STK type.STKs are the most common of the cytosolic kinases; i.e., kinases thatperform their function in that part of the cytoplasm other than thecytoplasmic organelles and cytoskelton. The cytosol is the region withinthe cell where much of the cell's intermediary metabolic andbiosynthetic activity occurs; e.g., it is in the cytosol that proteinsare synthesized on ribosomes.

RTKs, CTKs and STKs have all been implicated in a host of pathogenicconditions including, significantly, cancer. Other pathogenic conditionswhich have been associated with PTKs include, without limitation,psoriasis, hepatic cirrhosis, diabetes, angiogenesis, restenosis, oculardiseases, rheumatoid arthritis and other inflammatory disorders,immunological disorders such as autoimmune disease, cardiovasculardisease such as atherosclerosis and a variety of renal disorders.

With regard to cancer, two of the major hypotheses advanced to explainthe excessive cellular proliferation that drives tumor developmentrelate to functions known to be PK regulated. That is, it has beensuggested that malignant cell growth results from a breakdown in themechanisms that control cell division and/or differentiation. It hasbeen shown that the protein products of a number of proto-oncogenes areinvolved in the signal transduction pathways that regulate cell growthand differentiation. These protein products of proto-oncogenes includethe extracellular growth factors, transmembrane growth factor PTKreceptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussedabove.

In view of the apparent link between PK-related cellular activities andwide variety of human disorders, it is no surprise that a great deal ofeffort is being expended in an attempt to identify ways to modulate PKactivity. Some of this effort has involved biomimetic approaches usinglarge molecules patterned on those involved in the actual cellularprocesses (e.g., mutant ligands (U.S. Pat. No. 4,966,849); solublereceptors and antibodies (App. No. WO 94/10202, Kendall and Thomas,Proc. Nat'l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature,362:841-844 (1993)); RNA ligands (Jelinek, et al., Biochemistry,33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, etal., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys.,152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc.Am. Assoc. Cancer Res., 35:2268 (1994)).

In addition to the above, attempts have been made to identify smallmolecules which act as PK inhibitors. For example, bis-monocylic,bicyclic and heterocyclic aryl compounds (PCT WO 92/20642),vinylene-azaindole derivatives (PCT WO 94/14808) and1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have beendescribed as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No.5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No.5,302,606), quinazoline derivatives (EP App. No. 0 566 266 A1),selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxyliccompounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO91/15495) have all been described as PTK inhibitors useful in thetreatment of cancer.

SUMMARY OF THE INVENTION

Our own efforts to identify small organic molecules which modulate PKactivity and which, therefore, would be expected to be useful in thetreatment and prevention of disorders driven by abnormal PK activity,has led us to the discovery of a family of novel geometricallyrestricted 2-indolinone derivatives which exhibit PK modulating abilityand which are the subject of this invention.

Thus, the present invention relates generally to novel geometricallyrestricted 2-indolinone derivatives and their prodrugs andphysiologically acceptable salts which modulate the activity of receptortyrosine kinases (RTKs), non-receptor protein tyrosine kinases (CTKs)and serine/threonine protein kinases (STKs). In addition, the presentinvention relates to -the preparation and use of pharmaceuticalcompositions of the disclosed compounds and their physiologicallyacceptable salts and prodrugs in the treatment or prevention of PKdriven disorders such as, by way of example and not limitation, cancer,diabetes, hepatic cirrhosis, cardiovasacular disease such asatherosclerosis, angiogenesis, immunological disorders such asautoimmune disease and renal disease.

The terms “2-indolinone,” “indolin-2-one,” “2-oxindole” and “oxindole”are used interchangably herein; all refer to a chemical compound havingthe general structure:

As used herein, the above terms are deemed to include sulfur derivative;i.e., when Z=sulfur.

“Geometrically restricted” refers to the chemical structure about adouble bond wherein groups attached to the double bond are set in theirspatial relationship to one another by the very nature of the doublebond. That is, atoms attached to a double bond must be coplanar; i.e.,in the same plane as the atoms of the double bond itself. This is bestdemonstrated insofar as the compounds of this invention are concerned bylooking at the generic structures shown in Formulas 1 and 2:

Formula 1 represents a backbone structure of a compound of thisinvention. It is understood that 1 is presented by way of example onlyand not limitation; backbone structures other than that shown in 1 arewithin the scope and spirit of this invention. The point to be gleanedfrom 1 is the relationship of the atoms attached to the double bond atthe 3-position of the indolinone. In 1, ring system a and ring system bare linked to the double bond through a ring carbon. Since the atoms toeither side of the linking carbon atom must be coplanar due to thedouble bond, and since the rings themselves are internally co-planar,the entire molecule, ring systems a and b and the double bond, areco-planar. This is in contrast to Formula 2, wherein a single bondconnects ring system a′ to the double bond. Ring system a′ is thereforefree to rotate about the single bond permitting the ring systems a′ andb′ to be non-co-planar, potentially even being perpendicular to oneanother.

A “pharmacological composition” refers to a mixture of one or more ofthe compounds described herein, or physiologically acceptable saltsthereof, with other chemical components, such as physiologicallyacceptable carriers and/or excipients. The purpose of a pharmacologicalcomposition is to facilitate administration of a compound to anorganism.

As used herein, a “physiologically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

An “excipient” refers to an inert substance added to a pharmacologicalcomposition to further facilitate administration of a compound.Examples, without limitation, of excipients include calcium carbonate,calcium phosphate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils and polyethylene glycols.

A “prodrug” refers to an agent which is converted into the parent drugin vivo. Prodrugs are often useful because, in some situations, they maybe easier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent drug is not. Theprodrug may also have improved solubility in pharmacologicalcompositions over the parent drug. An example, without limitation, of aprodrug would be a compound of the present invention which isadministered as an ester (the “prodrug”) to facilitate transmittalacross a cell membrane where water solubility is detrimental to mobilitybut then is metabolically hydrolyzed to the carboxylic acid, the activeentity, once inside the cell where water solubility is beneficial.

A further example of a prodrug might be a short polypeptide, forexample, without limitation, a 2-10 amino acid polypeptide, bondedthrough a terminal amino group to a carboxy group of a compound of thisinvention wherein the polypeptide is hydrolyzed or metabolized in vivoto release the active molecule.

1. THE COMPOUNDS

General Structural Features.

In one aspect, the present invention relates to a geometricallyrestricted 2-indolinone compound having chemical structure I, II or III:

The scope of this invention includes physiologically acceptable saltsand prodrugs of the compound claimed herein.

R¹ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, hydroxy, alkoxy, —C(═O)OR″, R″C(═O)O—, C-amido,C-thioamido, acetyl, —S(═O)₂R″, and trihalomethylsulfonyl, wherein

R″ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,aryl, heteroaryl (bonded through a ring carbon), and heteroalicyclic(bonded through a ring carbon);

A, B, D and E are independently selected from the group consisting ofcarbon and nitrogen, wherein

when A, B, D or E is nitrogen, R², R³, R⁴ or R⁵, respectively, does notexist;

F, G, J and K are independently selected from the group consisting ofcarbon, nitrogen, oxygen and sulfur, wherein

when n is 1 and F, G, J or K is an atom other than carbon, R⁶, R⁷, R⁸ orR⁹, respectively, does not exist;

when n is 0 and F, G or K is oxygen or sulfur, R⁶, R⁸ or R⁹,respectively, does not exist.

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selected from thegroup consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, —S(═O)R″, —S(═O)₂R″, S-sulfonamido,N-sulfonamido, N-trihalo-methanesulfonamido, —C(═O)R″, —C(═O)OR″,R″C(═O)O—, cyano, nitro, halo, cyanato, isocyanato, thiocyanato,isothiocyanato, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, amino and —NR¹³R¹⁴;

R² and R³ or R³ and R⁴ or R⁴ and R⁵ or R⁶ and R⁷ or R⁷ and R⁸ or R⁸ andR⁹ may combine to form a methylenedioxy or an ethylenedioxy group;

R¹³ and R¹⁴ are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic,—C(═O)R″, acetyl, —S(O)₂R″, trihalomethane-sulfonyl and, combined, afive-member or a six-member heteroalicyclic ring;

R¹⁰, R¹¹ and R¹² are independently selected from the group consisting ofalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, cyano,trihalomethyl, hydroxy, alkoxy, alkylthio, aryloxy, arylthio, R″C(═O)O—,—C(═O)OR″, —C(═O)O⁻M⁺, —(CH₂)C(═O)OR″, —(CH₂)_(r)C(═O)O⁻M⁺, C-amido,N-amido, cyanato, isocyanato, thiocyanato, isothiocyanato, amino,—S(═O)R″, —S(═O)₂R″, nitro and —NR¹³R¹⁴;

R¹⁰ and R¹¹ or R¹¹ and R¹² may combine to form an endo double bond;

Z is selected from the group consisting of oxygen and sulfur; r is 1, 2,3, 4, 5, or 6; and,

n is 0 or 1.

The term “endo” refers to a double bond contained within a ringstructure; for example, the double bond in the following structure is an“endo” double bond:

As used herein, the term “alkyl” refers to a saturated aliphatichydrocarbon including straight chain and branched chain groups.Preferably, the alkyl group has 1 to 20 carbon atoms (whenever itappears herein, a numerical range such as “1 to 20” refers to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc. up to and including 20 carbon atoms). More preferably, it isa medium size alkyl having 1 to 10 carbon atoms. Most preferably, it isa lower alkyl having 1 to 4 carbon atoms. The alkyl group may besubstituted or unsubstituted. When substituted, the substituent group(s)is preferably one or more individually selected from cycloalkyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy,O-carboxy, cyanato, isocyanato, thiocyanato, isothiocyanato, nitro,silyl, amino and —NR¹³R¹⁴, R¹³ and R¹⁴ being as defined above.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone of more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,adamantane, cyclohexadiene, cycloheptane and, cycloheptatriene. Acycloalkyl group may be substituted or unsubstituted. When substituted,the substituent group(s) is preferably one or more individually selectedfrom alkyl, aryl, heteroaryl, heteroalycyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl,carboxy, O-carbamyl, N-carbamyl, C-amido, N-amido, nitro, amino and—NR¹³R¹⁴, with R¹³ and R¹⁴ being as defined above.

An “alkenyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbondouble bond.

An “alkynyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbontriple bond.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more selectedfrom halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy,O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalo-methanesulfonamido, amino and —NR¹³R ¹⁴, R¹³ and R¹⁴ being asdefined above.

As used herein, a “heteroaryl” group refers to a monocyclic or fusedring (i.e., rings which share an adjacent pair of atoms) group having inthe ring(s) one or more atoms selected from the group consisting ofnitrogen, oxygen and sulfur and, in addition, having a completelyconjugated pi-electron system. Examples, without limitation, ofheteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline,purine and carbazole. The heteroaryl group may be substituted orunsubstituted. When substituted, the substituted group(s) is preferablyone or more selected from alkyl, cycloalkyl, halo, trihalomethyl,hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro,carbonyl, thiocarbonyl, sulfonamido, carboxy, sulfinyl, sulfonyl,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, amino and —NR¹³R¹⁴, R¹³ and R¹⁴ being defined above.

A “heteroalicyclic” group refers to a monocyclic or fused ring grouphaving in the ring(s) one or more atoms selected from the groupconsisting of nitrogen, oxygen and sulfur. The rings may also have oneor more double bonds. However, the rings do not have a completelyconjugated pi-electron system. The heteroalicyclic ring may besubstituted or unsubstituted. When substituted, the substituted group(s)is preferably one or more selected from alkyl, cycloaklyl, halo,trihalomethyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio,cyano, nitro, carbonyl, thiocarbonyl, carboxy, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thio-carbamyl, sulfinyl, sulfonyl, C-amido, N-amido,amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ being as defined above.

A “hydroxy” group refers to an —OH group.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group,as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

A “mercapto” group refers to an —SH group.

A “alkylthio” group refers to both an S-alkyl and an —S-cycloalkylgroup, as defined herein.

A “arylthio” group refers to both an —S-aryl and an —S-heteroaryl group,as defined herein.

A “carbonyl” group refers to a —C(═O)R″ group, where R″ is selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl(bonded through a ring carbon) and heteroalicyclic (bonded through aring carbon), as defined herein.

An “aldehyde” group refers to a carbonyl group where R″ is hydrogen.

A “cycloketone” refer to a cycloalkyl group in which one of the carbonatoms which form the ring has a “═O” bonded to it; i.e. one of the ringcarbon atoms is a —C(═O)-group.

A “thiocarbonyl” group refers to a —C(═S)R″ group, with R″ as definedherein.

An “O-carboxy” group refers to a R″C(═O)O-group, with R″ as definedherein.

A “C-carboxy” group refers to a —C(═O)OR″ groups with R″ as definedherein.

As used herein, an “ester” is a C-carboxy group, as defined herein,wherein R″ is any of the listed groups other than hydrogen.

A “C-carboxy salt” refers to a —C(═O)O⁻M⁺ group wherein M⁺ is selectedfrom the group consisting of lithium, sodium, magnesium, calcium,potassium, barium, iron, zinc and quaternary ammonium.

An “acetyl” group refers to a —C(═O)CH₃ group.

A “carboxyalkyl” group refers to —(CH₂)_(r)C(═O)OR″ wherein r is 1-6 andR″ is as defined above.

A “carboxyalkyl salt” refers to a —(CH₂)_(r)C(═O)O⁻M⁺ wherein M⁺ isselected from the group consisting of lithium, sodium, potassium,calcium, magnesium, barium, iron, zinc and quaternary ammonium.

A “carboxylic acid” group refers to a C-carboxy group in which R″ ishydrogen.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “trihalomethyl” group refers to a —CX₃ group wherein X is a halo groupas defined herein.

A “trihalomethanesulfonyl” group refers to a X₃CS(═O)₂-group with X asdefined above.

A “cyano” group refers to a —C═—N group.

A “cyanato” group refers to a —CNO group.

An “isocyanato” group refers to a —NCO group.

A “thiocyanato” group refers to a —CNS group.

An “isothiocyanato” group refers to a —NCS group.

A “sulfinyl” group refers to a —S(═O)R″ group, with R″ as definedherein.

A “sulfonyl” group refers to a —S(═O)₂R″ group, with R″ as definedherein.

A “sulfonamido” group refers to a —S(═O)₂NR¹³R¹⁴, with R″ and R¹⁴ asdefined herein.

A “trihalomethanesulfonamido” group refers to a X₃CS(═O)₂NR¹³-group withX and R¹³ as defined herein.

An “O-carbamyl” group refers to a —OC(═O)NR¹³R¹⁴ group with R¹³ and R¹⁴as defined herein.

An “N-carbamyl” group refers to a R¹⁴OC(═O)NR¹³— group, with R¹³ and R¹⁴as defined herein.

An “O-thiocarbamyl” group refers to a —OC(═S)NR¹³R¹⁴ group with R¹³ andR¹⁴ as defined herein.

An “N-thiocarbamyl” group refers to a R¹⁴OC(═S)NR¹³— group, with R¹³ andR¹⁴ as defined herein.

An “amino” group refers to an —NR¹³R¹⁴ group, with R¹³ and R¹⁴ bothbeing hydrogen.

A “C-amido” group refers to a —C(═O)NR¹³R¹⁴ group with R¹³ and R¹⁴ asdefined herein. An “N-amido” group refers to a R¹³C(═O)NR¹⁴— group withR¹³ and R¹⁴ as defined herein.

A “nitro” group refers to a —NO₂ group.

A “quaternary ammonium” group refers to a —⁺NR¹³R¹⁴R¹⁵ group whereinR¹³R¹⁴ and R¹⁵ are independently selected from the group consisting ofhydrogen and unsubstituted lower alkyl.

A “methylenedioxy” group refers to a —OCH₂O— group wherein the oxygenatoms are bonded to adjacent ring carbon atoms.

An “ethylenedioxy” group refers to a —OCH₂CH₂O— group wherein the oxygenatoms are bonded to adjacent ring carbon atoms.

Another aspect of this invention is a combinatorial library of at least10 compounds formed by reacting an oxindole having the general chemicalstructure:

with a cycloketone having one of the general chemical structures:

wherein A, B, D, E, F, G, J, K, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹ and R¹² are as previously defined herein.

By “reacting” is meant placing one of the above oxindoles and one of theabove cycloketones in a chemical environment wherein they will interactwith on another to form a covalent bond between them. In the presentcase, the covalent bond will ultimately be a double bond from the carbonatom at the 3-position of the oxindole to the keto carbon atom of thecycloketone.

A “combinatorial library” refers to all the compounds formed by thereaction of each compound in one dimension of a multi-dimensional arraywith a compound in each of the other dimensions of the multi-dimensionalarray. As used herein, the multi-dimensional array is two-dimensional,one dimension being all the oxindoles of this invention, the otherdimension being all the cycloketones of this invention. Each oxindolemay be reacted with each of the cycloketones to form a 2-indolinone. All2-indolinone compounds formed in this manner are within the scope ofthis invention. Also within the scope of this invention are smallercombinatorial libraries formed by the reaction of some of the oxindolesof this invention with all of the cycloketones of this invention or allof the oxindoles with some of the cycloketones or some of the oxindoleswith some of the cycloketones.

It is another aspect of this invention that a combinatorial library maybe used to screen compounds of this invention for a desired activity.

By a “desired activity” is meant the ability to modulate the catalyticactivity of a selected protein kinase.

By “screening” is meant to contact an entire combinatorial library ofcompounds or any portion thereof with one or more target protein kinasesand then observe the effect of the compounds on the catalytic activityof the protein kinase.

Yet another aspect of this invention is a compound which modulatesprotein kinase activity, in particular RTK, CTK or STK kinase catalyticactivity.

Preferred Structural Features.

A presently preferred embodiment of this invention is a compound inwhich:

n is 1;

A, B, D and E are carbon;

R¹ is hydrogen; and,

Z is oxygen.

A further presently preferred embodiment of this invention is one inwhich:

n is 1;

A, B, D and E are carbon;

R¹ is hydrogen;

Z is oxygen; and,

F, G, J and K are carbon.

Still another presently preferred embodiment of this invention is acompound in which:

n is 0;

A, B, D and E are carbon; and,

Z is oxygen.

A compound in which:

n is 0;

A, B, D and E are carbon;

Z is oxygen;

F is nitrogen;

R⁹ is hydrogen; and,

G and K are carbon

is yet another presently preferred embodiment of this invention.

A presently preferred embodiment of this invention would also be acompound in which:

n is 0;

A, B, D and E are carbon;

Z is oxygen;

K is nitrogen;

R⁶ is hydrogen; and,

F and G are carbon.

A further presently preferred embodiment of this invention is a compoundin which one or two of F, G, J or K are independently nitrogen.

It is likewise a presently preferred embodiment of this invention that

n is 1;

A, B, D and E are carbon;

R¹ is hydrogen;

Z is oxygen;

R¹³ is hydrogen; and,

R¹⁴ is unsubstituted lower alkyl.

It is still another presently preferred embodiment of this inventionthat:

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selected from thegroup consisting of:

hydrogen;

unsubstituted lower alkyl;

lower alkyl substituted with a group selected from the group consistingof halo, —C(═O)OR″ and —NR¹³R¹⁴;

unsubstituted lower alkoxy;

lower alkoxy substituted with a group selected from the group consistingof halo, —C(═O)OR″, unsubstituted aryl or —NR¹³R¹⁴;

trihalomethyl;

unsubstituted alkenyl;

unsubstituted alkynyl;

unsubstituted aryl;

aryl substituted with one or more groups independently selected from thegroup consisting of unsubstituted lower alkyl or lower alkyl substitutedwith a group selected from the group consisting of halo, —C(═O)OR″ and—NR¹³R¹⁴;

unsubstituted heteroalicyclic;

heteroalicyclic substituted with one or more groups independentlyselected from the group consisting of unsubstituted lower alkyl,—C(═O)H, —C(═O)— (unsubstituted lower alkyl), hydroxy, unsubstitutedalkoxy, alkoxy substituted with a group selected from the groupconsisting of halo, —C(═O)OR″ and —NR¹³R¹⁴;

unsubstituted aryloxy;

aryloxy substituted with a group independently selected from the groupconsisting of unsubstituted lower alkyl, trihalomethyl, halo, hydroxyand amino;

mercapto;

unsubstituted alkylthio;

unsubstituted arylthio;

arylthio substituted with one or more groups independently selected fromthe group consisting of halo, hydroxy and amino;

S-sulfonamido;

—C)═O)OR″;

R″C(═O)O—;

hydroxy;

cyano;

nitro;

halo;

C-amido;

N-amido;

amino; and,

—NR¹³R¹⁴.

A compound in which:

n is 1;

A, B, D and E are carbon;

R¹ is hydrogen;

Z is oxygen;

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selected from thegroup consisting of:

hydrogen;

unsubstituted lower alkyl;

lower alkyl substituted with a group selected from the group consistingof halo, —C(═O)OR″ and —NR¹³R¹⁴;

unsubstituted lower alkoxy;

lower alkoxy substituted with a group selected from the group consistingof halo, —C(═O)OR″, unsubstituted aryl or —NR¹³R¹⁴;

trihalomethyl;

unsubstituted alkenyl;

unsubstituted alkynyl;

unsubstituted aryl;

aryl substituted with one or more groups independently selected from thegroups consisting of unsubstituted lower alkyl or lower alkylsubstituted with a group selected from the group consisting of halo,—C(═O)OR″ and —NR¹³R¹⁴;

unsubstituted heteroalicyclic;

heteroalicyclic substituted with one or more groups independentlyselected from the group consisting of unsubstituted lower alkyl,—C(═O)H, —C(═O)— (unsubstituted lower alkyl), hydroxy, unsubstitutedalkoxy, alkoxy substituted with a group selected from the groupconsisting of halo, —C(═O)OR″ and —NR¹³R¹⁴;

unsubstituted aryloxy;

aryloxy substituted with a one or more groups independently selectedfrom the group consisting of unsubstituted lower alkyl, trihalomethyl,halo, hydroxy and amino;

mercapto;

unsubstituted alkylthio;

unsubstituted arylthio;

arylthio substituted with one or more groups independently selected fromthe group consisting of halo, hydroxy or amino;

S-sulfonamido;

—C(═O)OR″;

R″C(═O)O—;

hydroxy;

cyano;

nitro;

halo;

C-amido;

N-amido;

amino; and,

—NR¹³R¹⁴, wherein

R¹³ is hydrogen and R¹⁴ is unsubstituted lower alkyl is anotherpresently preferred embodiment of this invention.

A still further presently preferred embodiment of this invention is acompound in which:

R¹⁰, R¹¹ and R¹² are independently selected from the group consisting ofhydrogen, unsubstituted lower alkyl, (CH₂)_(r)C(═O)OR″,—(CH₂)_(r)C(═O)O⁻M⁺, halo, hydroxy, alkoxy, R″C(═O)O—, —C(═O))OR″,—C(═O)O⁻M⁺, amino, C-amido, N-amido, nitro and —NR¹³R¹⁴.

A compound in which:

n is 1;

A, B, D and E are carbon;

R¹ is hydrogen;

Z is oxygen;

R¹³ is hydrogen;

R¹⁴ is unsubstituted lower alkyl; and,

R¹⁰, R¹¹ and R¹² are independently selected from the group consisting ofhydrogen, unsubstituted lower alkyl, —(CH₂)C(═O)OR″,—(CH₂)_(r)C(═O)O⁻M⁺, halo, hydroxy, alkoxy, R″C(═O)O—, —C(═O)OR″,—C(═O)O⁻M⁺, amino, C-amido, N-amido, nitro and —NR¹³R¹⁴ is also apresently preferred embodiment of this invention.

A compound having the structural features in the paragraph immediatelyabove wherein at least one of R¹⁰, R¹¹ or R¹² is selected from the groupconsisting of —C(═O)OR″, —C(═O)O⁻M⁺, —(CH₂)_(r)C(═O)OR″ and—(CH₂)_(r)C(═O)O⁻M⁺ is a presently preferred embodiment of thisinvention.

It is also a presently preferred embodiment of this invention that, in acompound having the structural features described in the paragraphimmediately above this one, r of the —(CH₂)_(r)C(═O)OR″ or—(CH₂)_(r)C(═O)O⁻ M⁺ group is 1 or 2.

Representative compounds of this invention are shown in Table 1. Thecompounds shown are presented by way of example only and are not to beconstrued as limiting the scope of this invention in any mannerwhatsoever.

2. BRIEF DESCRIPTION OF THE TABLES

1. Table 1 shows the chemical structures of exemplary compounds of thisinvention. The compound numbers correspond to the compound numbers inthe Examples section, below. That is, the synthesis of compound 1 inTable 1 is Example 1 in the Examples section. These compounds arepresented as examples only and are not to be construed as limiting thescope of this invention in any manner whatsoever.

2. Table 2 shows the results of biological assays of exemplary compoundsof this invention. As above, the compound numbers is Table 2 correspondto the compound numbers in Table 1. The bioassays used are described indetail below. The results are given in terms of IC₅₀, the micromolar(μM) concentration of the compound being tested which effects a 50%change in the activity of a target PTK compared to the activity of thePTK in a control in which no compound of this invention is present.Specifically, the results shown indicate the concentration of the testcompound needed to effect a 50% inhibition of the activity of the targetPTK observed in the absence of a compound of this invention.

3. Table 3 shows the results of in vivo tests using the A375 sc cellline in the animal xenograft model described below. Compound 13 has thechemical structure shown in Table 1.

TABLE 1 Compound No.: Structure Compound 1 

Compound 2 

Compound 3 

Compound 4 

Compound 5 

Compound 6 

Compound 7 

Compound 8 

Compound 9 

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

3. THE BIOCHEMISTRY

In yet another embodiment, this invention relates to a method for themodulation of the catalytic activity of PKs by contacting a PK with acompound of this invention or a physiologically acceptable salt orprodrug thereof.

A further embodiment of this invention is a method for identifying acompound which modulates the activity of a PK whichmethod consists ofcontacting a cell which the PK of interest with a compound andmonitoring the effect of the compound on the cell.

By “PK” is meant RTKs, CTKs and STKs; i.e., the modulation of RTK, CTKand STK catalyzed signaling processes are contemplated by thisinvention.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures by,practitioners of the chemical, pharmacological, biological, biochemicaland medical arts.

As used herein, the term “modulation” or “modulating” refers to thealteration of the catalytic activity of RTKs, CTKs and STKs. Inparticular, modulating refers to the activation of the catalyticactivity of RTKS, CTKs and STKS, preferably the activation or inhibitionof the catalytic activity of RTKs, CTKs and STKs, depending on theconcentration of the compound or salt to which the RTK, CTK or STK isexposed or, more preferably, the inhibition of the catalytic activity ofRTKs, CTKs and STKs.

The term “catalytic activity” as used herein refers to the rate ofphosphorylation of tyrosine under the influence, direct or indirect, ofRTKs and/or CTKs or the phosphorylation of serine and threonine underthe influence, direct or indirect, of STKs.

The term “contacting” as used herein refers to bringing a compound ofthis invention and a target PK together in such a manner that thecompound can affect the catalytic activity of the PK, either directly;i.e., by interacting with the kinase itself, or indirectly; i.e., byinteracting with another molecule on which the catalytic activity of thekinase is dependent. Such “contacting” can be accomplished in a testtube, a petri dish or the like. In a test tube, contacting may involveonly a compound and a PK of interest or it may involve whole cells.Cells may also be maintained or grown in cell culture dishes andcontacted with the compound in that environment. In this context, theability of a particular compound to affect a PK related disorder; i.e.,the IC₅₀, of the compound, defined below, can be determined before useof the compounds in vivo with more complex living organisms isattempted. For cells outside the organism, multiple methods exist, andare well-known to those skilled in the art, to get the PKs in contactwith the compounds including, but not limited to, direct cellmicroinjection and numerous transmembrane carrier techniques.

By “monitoring” or “observing” is meant detecting the effect ofcontacting a compound with a cell expressing a particular PK.

The detected effect can be a change in cell phenotype, in the catalyticactivity of a PK or a change in the interaction of a PK with a naturalbinding partner.

“Cell phenotype” refers to the outward appearance of a cell or tissue orthe biological function of the cell or tissue. Examples, withoutlimitation, of a cell phenotype is cell size, cell growth, celldifferentiation, cell proliferation, cell survival, apoptosis andnutrient uptake and use. Such phenotypic characteristics are detectableby techniques well-known in the art.

A “natural binding partner” refers to a polypeptide that binds to aparticular PK in a cell. Natural binding partners can plan a role inpropagating a signal in a PK-mediated signal transduction process. Achange in the interaction of the natural binding partner with the PK canmanifest itself as an increased or decreased concentration of thePK/natural binding partner complex resulting in a detectable change inthe ability of the PK to mediate signal transduction.

RTK mediated signal transduction is initiated by extracellularinteraction with a specific growth factor (ligand), followed by receptordimerization, transient stimulation of the intrinsic protein tyrosinekinase activity and phosphorylation. Binding sites are thereby createdfor intracellular signal transduction molecules and lead to theformation of complexes with a spectrum of cytoplasmic signalingmolecules that facilitate the appropriate cellular response (e.g., celldivision, metabolic effects on the extracellular microenvironment,etc.). See, Schlessinger and Ullrich, 1992, Neuron 9:303-391.

It has been shown that tyrosine phosphorylation sites on growth factorreceptors function as high-affinity binding sites for SH2 (src homology)domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423;Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785); Songyang et al.,1993, Cell 72:767-778; and Koch et al., 1991, Science 252:668-678.Several intracellular substrate proteins that associate with RTKs havebeen identified. They may be divided into two principal groups: (1)substrates which have a catalytic domain; and (2) substrates which lacksuch domain but serve as adapters and associate with catalyticallyactive molecules. Songyang et al., 1993, Cell, 72:767-778. Thespecificity of the interactions between receptors and SH2 domains oftheir substrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. Songyang et al., 1993, Cell, 72:767-778. Theseobservations suggest that the function of each RTK is determined notonly by its pattern of expression and ligand availability but also bythe array of downstream signal transduction pathways that are activatedby a particular receptor. Thus, phosphorylation provides an importantregulatory step which determines the selectivity of signaling pathwaysrecruited by specific growth factor receptors as well as differentiationfactor receptors.

STKs, being primarily cytosolic, affect the internal biochemistry of thecell, often as a down-line response to a PTK event. STKs have beenimplicated in the signaling process which initiates DNA synthesis andsubsequent mitosis leading to cell proliferation.

Thus, PK signal transduction results in, among other responses, cellproliferation, differentiation, growth and metabolism. Abnormal cellproliferation may result in a wide array of disorders and diseases,including the development of neoplasia such as carcinoma, sarcoma,glioblastoma and hemangioma, disorders such as leukemia, psoriasis,arteriosclerosis, arthritis and diabetic retinopathy and other disordersrelated to uncontrolled angiogenesis and/or vasculogenesis.

A precise understanding of the mechanism by which the compounds of thisinvention inhibit PKs is not required in order to practice the presentinvention. However, while not hereby being bound to any particularmechanism or theory, it is believed that the compounds interact with theamino acids in the catalytic region of PKs. PKs typically possess abi-lobate structure wherein ATP appears to bind in the cleft between thetwo lobes in a region where the amino acids are conserved among PKs.Inhibitors of PKs are believed to bind by non-covalent interactions suchas hydrogen bonding, van der Waals forces and ionic interactions in thesame general region where the aforesaid ATP binds to the PKs. Morespecifically, it is thought that the 2-indolinone component of thecompounds of this invention binds in the general space normally occupiedby the adenine ring of ATP. Specificity of a particular molecule for aparticular PK may then arise as the result of additional interactionsbetween the various substituents on the 2-indolinone core and the aminoacid domains specific to particular PKs. Thus, different indolinonesubstituents may contribute to preferential binding to particular PKs.The ability to select compounds active at different ATP (or othernucleotide) binding sites makes the compounds of this invention usefulfor targeting any protein with such a site; i.e., not only PKs butprotein phosphatases as well. The compounds disclosed herein may thushave utility as in vitro assays for such proteins as well as exhibitingin vivo therapeutic effects through interaction with such proteins.

In another aspect, the protein kinase, the catalytic activity of whichis modulated by contact with a compound of this invention, is a proteintyrosine kinase, more particularly, a receptor protein tyrosine kinase.Among the receptor protein tyrosine kinases whose catalytic activity canbe modulated with a compound of this invention, or salt thereof, are,without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα,PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R,FGFR-2R, FGFR-3R and FGFR-4R.

The protein tyrosine kinase whose catalytic activity is modulated bycontact with a compound of this invention, or a salt or a prodrugthereof, can also be a non-receptor or cellular protein tyrosine kinase(CTK). Thus, the catalytic activity of CTKs such as, without limitation,Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn,Lck, Blk, Hck, Fgr and Yrk, may be modulated by contact with a compoundor salt of this invention.

Still another group of PKs which may have their catalytic activitymodulated by contact with a compound of this invention are theserine-threonine protein kinases such as, without limitation, CDK2 andRaf.

In another aspect, this invention relates to a method for treating orpreventing a PK related disorder by administering a therapeuticallyeffective amount of a compound of this invention, or a salt or a prodrugthereof, to an organism.

In a further aspect, this invention relates to a method for treating orpreventing a PK related disorder administering a therapeuticallyeffective amount of a pharmacological composition of a compound of thisinvention, or a salt or prodrug thereof, to an organism.

As used herein, “PK related disorder,” “PK driven disorder,” and“abnormal PK activity” all refer to a condition characterized byinappropriate; i.e., under or, more commonly, over, PK catalyticactivity, where the particular PK can be an RTK, a CTK or an STK.Inappropriate catalytic activity can arise as the result of either: (1)PK expression in cells which normally do not express PKs; (2) increasedPK expression leading to unwanted cell proliferation, differentiationand/or growth; or, (3) decreased PK expression leading to unwantedreductions in cell proliferation, differentiation and/or growth.Over-activity of PKs refers to either amplification of the gene encodinga particular PK or production of a level of PK activity which cancorrelate with a cell proliferation, differentiation and/or growthdisorder (that is, as the level of the PK increases, the severity of oneor more of the symptoms of the cellular disorder increases).Underactivity is, of course, the converse, wherein the severity of oneor more symptoms of a cellular disorder increase as the level of the PKdecreases.

As used herein, the terms “prevent”, “preventing” and “prevention” referto a method for barring an organism from acquiring a PK mediatedcellular disorder in the first place.

As used herein, the terms “treat”, “treating” and “treatment” refer to amethod of alleviating or abrogating a PK mediated cellular disorderand/or its attendant symptoms. With regard particularly to cancer, theseterms simply mean that the life expectancy of an individual affectedwith a cancer will be increased or that one or more of the symptoms ofthe disease will be reduced.

The term “organism” refers to any living entity comprised of at leastone cell. A living organism can be as simple as, for example, a singleeukariotic cell or as complex as a mammal, including a human being.

The term “therapeutically effective amount” as used herein refers tothat amount of the compound being administered which will relieve tosome extent one or more of the symptoms of the disorder being treated.In reference to the treatment of cancer, a therapeutically effectiveamount refers to that amount which has the effect of (1) reducing thesize of the tumor; (2) inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis; (3) inhibiting to some extent(that is, slowing to some extent, preferably stopping) tumor growth;and/or, (4) relieving to some extent (or, preferably, eliminating) oneor more symptoms associated with the cancer.

This invention is therefore directed to compounds which modulate PKsignal transduction by affecting the enzymatic activity of RTKs, CTKsand/or STKs, thereby interfering with the signals transduced by suchproteins. More particularly, the present invention is directed tocompounds which modulate RTK, CTK and/or STK mediated signaltransduction pathways as a therapeutic approach to cure many kinds ofsolid tumors, including but not limited to carcinomas, sarcomas,including Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma,astrocytoma, melanoma and myoblastoma. Treatment or prevention ofnon-solid tumor cancers such as leukemia are also contemplated by thisinvention. Indications may include, but are not limited to braincancers, bladder cancers, ovarian cancers, gastric cancers, pancreascancers, colon cancers, blood cancers, lung cancers and bone cancers.

Further examples, without limitation, of the types of disorders relatedto unregulated PK activity that the compounds described herein may beuseful in preventing, treating and studying, are cell proliferativedisorders, fibrotic disorders and metabolic disorders.

Cell proliferative disorders, which may be prevented, treated or furtherstudied by the present invention include cancer, blood vesselproliferative disorders and mesangial cell proliferative disorders.

Blood vessel proliferative disorders refer to disorders related toabnormal vasculogenesis (blood vessel formation) and angiogenesis(spreading of blood vessels). While vasculogenesis and angiogenesis playimportant roles in a variety of normal physiological processes such asembryonic development, corpus luteum formation, wound healing and organregeneration, they also play a pivotal role in cancer development wherethey result in the formation of new capillaries needed to keep a tumoralive. Other examples of blood vessel proliferation disorders includearthritis, where new capillary blood vessels invade the joint anddestroy cartilage, and ocular diseases, like diabetic retinopathy, wherenew capillaries in the retina invade the vitreous, bleed and causeblindness.

Conversely, disorders related to the shrinkage, contraction or closingof blood vessels, such as restenosis, may also be treated or preventedby the methods of this invention.

Fibrotic disorders refer to the abnormal formation of extracellularmatrices. Examples of fibrotic disorders include hepatic cirrhosis andmesangial cell proliferative disorders. Hepatic cirrhosis ischaracterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. An increased extracellularmatrix resulting in a hepatic scar can also be caused by viral infectionsuch as hepatitis. Lipocytes appear to play a major role in hepaticcirrhosis. Another fibrotic disorder is atherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial proliferativedisorders include various human renal diseases, such asglomerulonephritis, diabetic nephropathy and malignant nephrosclerosisas well such disorders as thrombotic microangiopathy syndromes,transplant rejection, and glomerulopathies. PDGFR has been implicated inthe maintenance of mesangial cell proliferation. Floege et al., 1993,Kidney International 43:47Sa54S.

As noted previously, PKs have been associated with cell proliferativedisorders. Thus it is not surprising that some members of the RTK familyhave been associated with the development of cancer. Some of thesereceptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233; Torpet al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science244:707-712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633) areover-expressed in many tumors and/or persistently activated by autocrineloops. In fact, in the most common and severe cancers these receptorover-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol.Sci., 111:119-133; Dickson et al., 1992, Cancer Treatment Res.61:249-273; Korc et al., 1992, J. Clin. Invest. 90:1352-1360) andautocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-1070;Korc et al., supra; Akbasak and Suner-Akbasak et al., supra) have beendemonstrated. For example, EGFR has been associated with squamous cellcarcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancerand bladder cancer. HER2 has been associated.with breast, ovarian,gastric, lung, pancreas and bladder cancer. PDGFR has been associatedwith glioblastoma and melanoma as well as lung, ovarian and prostatecancer. The RTK c-met has been associated with malignant tumorformation. For example, c-met has been associated with, among othercancers, colorectal, thyroid, pancreatic, gastric and hepatocellularcarcinomas and lymphomas. Additionally c-met has been linked toleukemia. Over-expression of the c-met gene has also been detected inpatients with Hodgkins disease and Burkitts disease.

Flk/KDR has likewise been associated with a broad spectrum of tumorsincluding, without limitation, mammary, ovarian and lung tumors as wellas gliomas such as glioblastoma.

IGF-IR, in addition to being implicated in nutritional support and intype-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). Inaddition, IGF-I, while integrally involved in the normal growth anddifferentiation of the nervous system, also appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475-2478. The importance of IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stemcells of the bone marrow)) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression, 1:301-326. In a series ofrecent publications, Baserga suggests that IGF-IR plays a central rolein the mechanism of transformation and, as such, could be a preferredtarget for therapeutic interventions for a broad spectrum of humanmalignancies. Baserga, 1995, Cancer Res., 55:249-252; Baserga, 1994,Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol., 14:4588-4595.

STKs have been implicated in many types of cancer including, notably,breast cancer (Cance, et al., Int. J. Cancer, 54:571-77 (1993)).

The association between abnormal PK activity and disease is notrestricted to cancer. For example, RTKs have been associated withdiseases such as psoriasis, diabetes mellitus, endometriosis,angiogenesis, atheromatous plaque development, Alzheimer's disease,epidermal hyperproliferation, neuro-degenerative diseases, age-relatedmacular degeneration and hemangiomas. For instance, EGFR is indicated incorneal and dermal wound healing. Defects in Insulin-R and IGF-1R areindicated in type-II diabetes mellitus. A more complete correlationbetween specific RTKs and their therapeutic indications is set forth inPlowman et al., 1994, DN&P, 7:334-339.

As noted previously, not only RTKs but CTKs as well including, but notlimited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk(reviewed by Bolen et al., 1992, FASEB J., 6:3403-3409) are involved inthe proliferative and metabolic signal transduction pathway and thuscould be expected, and have been shown, to be involved in manyPTK-mediated disorders to which the present invention is directed. Forexample, mutated src (v-src) has been demonstrated to be an oncoprotein(pp60^(v-src)) in chicken. Moreover, its cellular homolog, theproto-oncogene pp60^(c-src) transmits oncogenic signals of manyreceptors. Over-expression of EGFR or HER2/neu in tumors leads to theconstitutive activation of pp60^(cXsrc), which is characteristic ofmalignant cells but absent in normal cells. On the other hand, micedeficient in the expression of c-src exhibit an osteopetrotic phenotype,indicating a key participation of c-src in osteoclast function and apossible involvement in related disorders.

Similarly, Zap70 has been connected with T-cell signaling which may haveimplications in autoimmune disorders.

STKs have been associated with inflamation, autoimmune disease,immunoresponses, and hyperproliferation disorders such as restenosis,fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.

PKs have also been implicated in embryo implantation. Thus, thecompounds of this invention may provide an effective method ofpreventing such embryo implantation and thereby be useful as birthcontrol agents.

Finally, both RTKs and CTKs are currently suspected as being involved inhyperimmune disorders. Thus, it is an aspect of this invention thatprotein kinase related cancers such as, without limitation, squamouscell carcinoma, astrocytoma, Kaposi's sarcoma, glioblastoma, lungcancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer,prostate cancer, breast cancer, small-cell lung cancer, glioma,colorectal cancer, genitourinary cancer and gasterointestinal cancer maybe treated or prevented by administration to an organism of atherapeutically effective amount of a compound of this invention.

In a further aspect of this invention, non-cancer protein kinase relateddiorders such as, without limitation, diabetes, autoimmune disorder,immunological disorder, hyperproliferative disorder, restenosis,fibrosis, psoriasis, von Hippel-Lindau disease, osteoarthritis,rheumatoid arthritis, angiogenesis, inflammatory disorder andcardiovascular disease, may also be treated or prevented by theadministration of a therapeutically effective amount of a compound ofthis invention to an organism.

Tables 2 and 3 show the activity of representative compounds of thisinvention against several of the above-described RTKs. Neither thecompounds shown, the levels of activity indicated nor the specific RTKsaffected are to be construed as limiting the scope of this invention inany manner whatsoever.

TABLE 2 bio EGFR bio cell VEGF Compound (Page 5) PDGFR Pyk2 cell PDGFRbio Flk EGFR cell IGF bio FGF bio SRC HUVEC 1 >100 >100 >100 54.2 18.4DNH DNH 2 >100 >100 DNH >100 8.2 DNH DNH 3 >100 86.9 DNH(56% 30.2 2.6DNH DNH Inh) 4 >100 >100 DNH >100 DNH DNH 5 >100 50.8 79.3 10.9 3.1DNH >100 6 >100 5.5 8.1 2.5 96.7% Inh >100 7 >100 >100 DNH >100 7.1DNH >100 8 >100 >100 DNH >100 4.3 DNH 9 >100 >100 >100 2.9 DNH 10  >10063.7 >100 1.4 >100 >100 56.8 >100 0.33 11  >100 1.5 9.3 >1000.02 >100 >100 0.2 0.05 12   >25 >25 DNH >25 >25 >25 >25 >25 13  >1001.0, 3.2 4.5 >100 >100 >100 14  DNH = did not hit in primary assay

TABLE 3 Average Tumor Volume % Treatment (mm3) inhibition P value %Vehicle 756.1 — — 40 Compound 13 @ 200 mg/kg/day 278.8 63.1 >0.05 40Compound 13 @ 100 mg/kg/day 324.4 57.1 >0.05 10 Compound 13 @ 50mg/kg/day 569.9 24.6 >0.05 20

4. PHARMACOLOGICAL COMPOSITIONS AND THERAPEUTIC APPLICATIONS

A compound of the present invention, a prodrug thereto or aphysiologically acceptable salt of either the compound or its prodrug,can be administered as such to a human patient or in pharmacologicalcompositions in which the foregoing materials are mixed with suitablecarriers or excipient(s). Techniques for formulation and administrationof drugs may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition.

Routes of Administration.

As used herein, “administer” or “administration” refers to the deliveryof a compound, salt or prodrug of the present invention or of apharamacological composition containing a compound, salt or prodrug ofthis invention to an organism for the purpose of prevention or treatmentof a PK-related disorder.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

Composition/Formulation.

Pharmacological compositions of the present invention may bemanufactured by processes well known in the art; e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmacological compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.

For transmucosal administration, penetrants appropriate to the barrierto be permeated ate used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, lozenges, dragees, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient. Pharmacological preparations for oral use can be made using asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding other suitableauxiliaries if desired, to obtain tablets or dragee cores. Usefulexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch and potato starchand other materials such as gelatin, gum tragacanth, methyl cellulose,hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginicacid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmacological compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. Stabilizers may be added in these formulations, also.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray using a pressurized pack or a nebulizer and a suitable propellant,e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichloro tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds may also be formulated for parenteral administration,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating materials such assuspending, stabilizing and/or dispersing agents.

Pharmacological compositions for parenteral administration includeaqueous solutions of a water soluble form, such as, without limitation,a salt, of the active compound. Additionally, suspensions of the activecompounds may be prepared in a lipophilic vehicle. Suitable lipophilicvehicles include fatty oils such as sesame oil, synthetic fatty acidesters such as ethyl oleate and triglycerides, or materials such asliposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers and/or agents which increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions. Alternatively, the active ingredient may be in powder formfor constitution with a suitable vehicle, e.g., sterile, pyrogen-freewater, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as depot preparations. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. A compound of thisinvention may be formulated for this route of administration withsuitable polymeric or hydrophobic materials (for instance, in anemulsion with a pharamcologically acceptable oil), with ion exchangeresins, or as a sparingly soluble derivative such as, withoutlimitation, a sparingly soluble salt.

The pharmacological compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the PK modulating compounds of the invention may be provided asphysiologically acceptable salts wherein the claimed compound may formthe negatively or the positively charged species. Examples of salts inwhich the compound forms the positively charged moiety include, withoutlimitation, quaternary ammonium (defined elsewhere herein), salts suchas the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate,succinate wherein the nitrogen atom of the quaternary ammonium group isa nitrogen of the selected compound of this invention which has reactedwith the appropriate acid. Salts in which a compound of this inventionforms the negatively charged species include, without limitation, thesodium, potassium, calcium and magnesium salts formed by the reaction ofa carboxylic acid group in the compound with an appropriate base (e.g.sodium hydroxide (NaOH), potassium hydroxide (KOH), Calcium hydroxide(Ca(OH)₂), etc.).

Dosage.

Pharmacological compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose; i.e., the modulationof PK activity or the treatment or prevention of a PK-related disorder.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. Then, the dosage can be formulated for use inanimal models so as to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture (i.e., the concentrationof the test compound which achieves a half-maximal inhibition of the PKactivity). Such information can then be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (bothof which are discussed elsewhere herein) for a subject compound. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active specie which are sufficient to maintain thekinase modulating effects. These plasma levels are referred to asminimal effective concentrations (MECs). The MEC will vary for eachcompound but can be estimated from in vitro data; e.g., theconcentration necessary to achieve 50-90% inhibition of a kinase may beascertained using the assays described herein. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration and other procedures known in the art may be employed todetermine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, etc.

Packaging.

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor of human or veterinary administration. Such notice, for example, maybe of the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition. Suitableconditions indicated on the label may include treatment of a tumor,inhibition of angiogenesis, treatment of fibrosis, diabetes, and thelike.

5. SYNTHESIS

The compounds of this invention, as well as the precursor 2-oxindolesand cycloketones, may be synthesized using the procedure describedbelow. Other approaches to the synthesis of the compounds and/orprecursors to the compounds of this invention may become apparent tothose skilled in the art based on the disclosures herein. Such alternateprocedures are within the scope and spirit of this invention.

General Synthetic Procedure.

One to three equivalents of the appropriate 2-oxindole and oneequivalent of the appropriate cycloketone are mixed together in water oran organic solvent such as, without limitation, C₁-C₁₀ alcohols(methanol, ethanol, octanol, etc.), ethylene glycol, cellosolve, diethylether, dichloromethane, chloroform, carbon tetrachloride, benzene,toluene, xylene, tetrahydrofuran, acetonitrile, dioxane,dimethylsulfoxide, dimethylformamide, dimethylacetamide,1-methyl-2-pyrrolidine, pyridine and the like. Preferably the solvent isan organic solvent selected from the group consisting ofdimethylsulfoxide, dimethylformamide, dimethylacetamide andN-methylpyrrolidine. Most preferably, the solvent is selected fromdimethylformamide and dimethylacetamide. A molar excess of an inorganicor organic base is added. The base may be, without limitation, sodiumhydroxide, potassium hydroxide, ammonium hydroxide, sodium methoxide,sodium ethoxide, potassium t-butoxide, triethylamine, triethanolamine,piperidine, morpholine, pyrrolidine and the like. Preferably, the baseis an organic base selected from the group consisting of triethylamine,piperidine, morpholine and pyrrolidine. Most preferably, the base ispiperidine. The resulting solution or mixture is stirred at from about20° C. to about 200° C. for from about 0.5 hour to about 100 hours atatmospheric pressure or in a sealed tube. The temperature is preferablyabout 90° C. to about 175° C., most preferably about 110° C. to about150° C. The reaction time is preferably 1 hour to about 72 hours. Themixture is then brought to room temperature. Dilute aqueous inorganicacid (for example, without limitation, 1N hydrochloric acid or 1Nsulfuric acid) is then added in an amount sufficient to neutralize thebase. The resulting mixture is extracted with a water insoluble organicsolvent such as without limitation, methylene chloride, diethyl ether,hexane, ethyl acetate, benzene or mixtures of solvents such as ethylacetate/hexane. Preferably, the organic solvent is ethyl acetate. Theorganic solvent layer is separated, dried and evaporated to give thecompound of this invention.

SPECIFIC SYNTHESES

The exemplary syntheses which follow are not to be construed as limitingthe scope of this invention in any manner.

EXAMPLE 1 3-(3-Methylindanl-ylidene)-1,3-dihydroindol-2-one

A mixture of 0.5 g of 3-methyl-1-indanone, 1.37 g oxindole and 5 mlpiperidine in 3 ml of dimethylforamide was heated in a sealed tube at130° C. overnight to yield a reddish-brown suspension. The mixture wasadded to 1N hydrochloric acid solution and extracted with ethyl acetate.The organic layer was washed with brine, dried with magnesium sulfateand concentrated. Chromatography afforded 120 mg of3-(3-methylindan-1-ylidene)-1,3-dihydroindol-2-one as an orange solid.

¹HNMR (360 MHz, DMSO-d6) δ: 10.54 (s, 1H, NH), 9.43 (d, J=9.7 Hz, 1H,Ar—H), 7.58 (d, J=9.36 Hz, 1H, Ar—H), 7.46 (d, J=4.68 Hz, 2H, Ar—H), 7.3(m, 1H, Ar—H), 7.19 (m, J=9.0 and 9.7 Hz, 1H, Ar—H), 6.99 (m, J=9.0 and9.36 Hz, 1H, Ar—H), 6.83 (m, J=9.0 and 1.08 Hz, 1H, Ar—H), 3.61 (dd,J=9.0 and 21.9 HZ 1H, 1×CH₂CH), 3.43 (m, 1H, CHCH₃), 2.86 (dd, J=4.3 and21.9 Hz, 1H, 1×CH₂CH), 1.35 (d, J=8.6 Hz, 3H, CH₃). MS (m/z (relativeintensity %, ion)): found 262.0 (100, [M+1]⁺); calc. 261.3.

EXAMPLE 2 3-(4-Methylindan-1-ylidene)-1,3-dihydroindol-2-one

A mixture of 0.5 g 4-methyl-1-indanone, 1.37 g oxindole and 3 mlpiperidine in 5 ml dimethylforamide was heated in a sealed tube at 130°C. for 3 days to yield a reddish-black suspension. The mixture was addedto 1N hydrochloric acid solution and extracted with ethyl acetate. Theorganic layer was washed with brine dried over magnesium sulfate andconcentrated. The resulting solid was precipitated from ethylacetate/hexanes (2×) followed by methylene chloride/hexanes to afford400 mg of 3-(4-methyl-indan-1-ylidene)-1,3-dihydroindol-2-one as ayellow solid.

¹HNMR (360 MHz, DMSO-d6) δ: 10.51 (s, 1H, NH), 9.38 (d, J=8.64 Hz, 1H,Ar—H), 7.58 (d, J=9.36 Hz, 1H, Ar—H), 7.2 (m, 3H, Ar—H), 6.99 (m, J=9.0and 9.36 Hz, 1H, Ar—H), 6.83 (d, J=9.0, 1H, Ar—H), 3.3 (m, 2H, 2×CH₂),3.0 (m, 2H, 2×CH₂), 2.29 (s, 3H, CH₃). MS (m/z (relative intensity %,ion)): found 262.0 (100, [M+1]⁺); calc. 261.3.

EXAMPLE 3 3-(5-Methoxyindan-1-ylidene)-1,3-dihydroindol-2-one

A mixture of 0.5 g 5-methoxy-1-indanone, 1.23 g oxindole and 2.7 mlpiperidine in 4 ml dimethylforamide was heated in a sealed tube at 130°C. for 3 days to yield a greenish-black suspension. The mixture wasadded to 1N hydrochloric acid solution and extracted with ethyl acetate.The organic layer was washed with brine, dried over magnesium sulfateand concentrated. Chromatography afforded 20 mg of3-(5-methoxyindan-1-ylidene)-1,3-dihydroindol-2-one as a brown solid.

¹HNMR (360 MHz, DMSO-d6) δ: 10.46 (s, 1H, NH), 9.52 (d, J=10.44 Hz, 1H,Ar—H), 7.53 (d, J=9.36 Hz, 1H, Ar—H), 7.15 (m, J=9.36 Hz, 1H, Ar—H), 7.0(m, 1H, Ar—H), 6.97 (m, J=9.0 and 9.36 Hz, 1H, Ar—H), 6.89 (dd, J=2.88and 10.44 Hz, 1H, Ar—H), 6.82 (m, J=9.0 Hz, 1H, Ar—H), 3.8 (s, 3H,OCH₃), 3.33 (m, 2H, 2×CH₂), 3.15 (m, 2H, 2×CH₂). MS (m/z (relativeintensity %, ion)): found 277.9 (100, M⁺); calc. 277.3.

EXAMPLE 4 3-(6-Methoxyindan-1-ylidene)-1,3-dihydroindol-2-one

A mixture of 0.5 g 6-methoxy-1-indanone, 1.23 g oxindole and 2.7 mlpiperidine in 4 ml of dimethylforamide was heated in a sealed tube 130°C. for 3 days to yield a greenish-black suspension. The mixture wasadded to 1N hydrochloric acid solution and extracted with ethyl acetate.The organic layer was washed with brine, dried over magnesium sulfateand concentrated. The resulting solid was precipitated from methylenechloride/hexanes (3×) to afford 290 mg3-(6-methoxyindan-1-ylidene)-1,3-dihydroindol-2-one as a yellowish-brownsolid.

¹NMR (360 MHz, DMSO-d6) δ: 10.51 (s, 1H, NH), 9.3 (s, 1H, Ar—H), 7.57(d, J=9.36 Hz, 1H, Ar—H), 7.35 (d, J 10.08 Hz, 1H, Ar—H), 7.19 (m, J=9.0and 9.36 Hz, 1H, Ar—H), 7.05 (dd, J=2.88 and 9.36 Hz, 1H, Ar—H), 6.99(m, J=8.64 and 9.0 Hz, 1H, Ar—H), 6.84 (m, J=8.64 Hz, 1H, Ar—H), 3.79(s, 3H, OCH₃), 3.35 (m, 2H, 2×CH₂), 3.10 (m, 2H, 2×CH₂). MS (m/z(relative intensity %, ion)): found 277.9 (100, M⁺); calc. 277.3.

EXAMPLE 5 3-(5-Dimethylaminoindan-1-ylidene)-1,3-dihydroindol-2-one

A mixture of 0.5 g 5-dimethylamino-1-indanone, 1.1 g oxindole and 2.5 mlpiperidine in 4 ml dimethylforamide was heated in a sealed tube at 130°C. for 12 hours to yield a reddish-black suspension. The mixture wasadded to 1N hydrochloric acid solution and extracted with ethyl acetate.The organic layer was washed with brine, dried with magnesium sulfateand concentrated. Chromatography afforded 35 mg of3-(5-dimethylamino-indan-1-ylidene)-1,3-dihydroindol-2-one as a brownsolid.

¹HNMR (360 MHz, DMSO-d6) δ: 10.28 (s, 1H, NH), 9.44. (d, J=10.08 Hz, 1H,Ar—H), 7.49 (d, J=7.56 Hz, 1H, Ar—H), 7.16 (t, J=8.28 and 7.56 Hz, 1H,Ar—H), 7.08 (m, 2H, Ar—H), 6.79 (d, J=7.92 Hz, 1H, Ar—H), 3.08 (m, 2H,2×CH₂), 3.03 (s, 3H, CH₃), 2.75 (m, 2H, 2×CH₂). MS (m/z (relativeintensity %, ion)): found 291.1 (100, [M+1]⁺); calc. 290.4.

EXAMPLE 6 3-(5-Aminoindan-1-ylidene)-1,3-dihydroindol-2-one

A mixture of 0.5 g 5-amino-1-indanone, 1.3 g oxindole and 3 mlpiperidine in 5 ml of dimethylforamide was heated in a sealed tube at130° C. for 3 days to yield a reddish-black suspension. The mixture wasadded to 1N hydrochloric acid and extracted with ethyl acetate. Theorganic layer was washed with brine, dried with magnesium sulfate andconcentrated. Chromatography afforded 45 mg of3-(5-aminoindan-1-ylidene)-1,3-dihydroindol-2-one as a brown solid.

¹HNMR (360 MHz, DMSO-d6) δ: 10.25 (s, 1H, NH), 9.34 (d, J=9.0 Hz, 1H,Ar—H), 7.48 (d, J=7.92 Hz, 1H, Ar—H), 7.07. (t, J=7.56 and 7.92 Hz, 1H,Ar—H), 6.92 (t, J=7.56 and 7.92 Hz, 1H, Ar—H), 6.79 (d, J=7.92 Hz, 1H,Ar—H), 6.5 (m, 2H, Ar—H), 6.0 (s, 2H, Ar—H), 3.25 (m, under water peak,CH₂), 3.01 (m, 2H, 2×CH₂). MS: (m/z (relative intensity %, ion)): found263.2 (35, [M+1]⁺); calc. 262.3.

EXAMPLE 7 3-(5-Chloroindan-1-ylidene)-1,3-dihydroindol-2-one

A mixture of 500 mg 5-chloro-1-indanone, 1.33 g oxindole and 0.5 mlpiperidine in 3 ml dimethylforamide was heated in a sealed tube at 95°C. overnight. Water was added to the reaction mixture to yield.an oilysolid. The mixture was sonicated for a few minutes then decanted. Thiswashing procedure was repeated several times. The solid was thenfiltered and washed with ethyl acetate/hexanes (1:3) to yield 172 mg of3-(5-chloroindan-1-ylidene)-1,3-dihydroindol-2-one as a mustard coloredsolid.

¹HNMR (360 MHz, DMSO-d6) δ: 10.56 (s, br, 1H, NH-1), 9.55 (d, J=8.90 Hz,1H), 7.57 (d, J=7.60 Hz, 1H, H-4), 7.52 (s, br, 1H), 7.37 (dd, J=1.76,8.90 Hz, 1H), 7.21 (t, J=7.76 Hz, 1H, H-6), 7.01 (d, J=7.60 Hz, 1H,H-5), 6.85 (d, J=7.76 Hz, 1H, H-7), 3.35-3.38 (m, 2H), and 3.18-3.23 (m,2H); MS (m/z (relative intensity %, ion)): 382 (100, [M+1]⁺).

EXAMPLE 8 3-(5-Piperidin-1-yl-indan-1-ylidene)-1,3-dihydroindol-2-one

A mixture of 0.5 g 5-fluoro-1-indanone, 13 g oxindole and 1 mlpiperidine in 4 ml dimethylforamide was heated in a sealed tube at 125°C. for 2.5 hours. The reaction mixture was poured into water and theaqueous layer was decanted. Ethyl acetate was then added slowly until asolid formed. The solid was filtered and washed with ethylacetate/hexanes to yield a curry colored solid. The solid was thenfurther purified by washing with dimethylforamide/acetone to give 122 mgof 3-(5-piperidin-1-yl-indan-1-ylidene)-1,3-dihydroindol-2-one as anorange solid.

¹HNMR (360 MHz, DMSO-d6) δ: 10.32 (s, 1H, NH-1), 9.42 (d, J=10.11 Hz, 1H), 7.50 (d, J=7.95 Hz, 1H, H-4), 7.10 (t, J=7.94 Hz, 2H, H-6), 6.93 (dt,J=1.08, 7.94 Hz, 1H, H-5), 6.88-6.86 (m, 2H), 6.80 (d, J=7.22 Hz, H-7),3.38 (br, m, 4H), 3.26-3.30 (m, 3H), 3.10-3.06 (m, 2H), and 1.59 (m, br,5H); MS (m/z (relative intensity, %, ion)): 331 (100, [M+1]⁺).

EXAMPLE 9 3-(5-Bromoindan-1-ylidene)-1,3-dihydroindol-2-one

A reaction mixture of 0.422 g 5-bromoindanone, 1.3 g oxindole and 0.9 mlpiperidine in 3 ml of dimethylforamide was heated in a sealed tube at110° C. overnight. The reaction mixture was poured into ice water andextracted with ethyl acetate. The organic layer was washed with brine,dried over anhydrous sodium sulfate and concentrated. The black residuewas chromatographed on a silica gel column, eluting with ethyl acetateand hexane to give 73.0 mg of3-(5-bromoindan-1-ylidene)-1,3-dihydroindol-2-one as a yellow solid.

¹HNMR (360 MHz, DMSO-d6) δ: 10.56 (s, 1H, NH-1), 9.46 (d, J=9.20 Hz,1H), 7.67 (s, 1H), 7.56 (d, J=7.84 Hz, 1H, H-4), 7.50 (d, J=9.20 Hz,1H), 7.21 (t, J=7.84 Hz, 1H, H-6), 7.00 (t, J=7.61 Hz, 1H, H-5), 6.83(d, J=7.61 Hz, 1H, H-7), 3.33-3.36 (m, 2H, CH₂), and 3.17-3.20 (m, 2H,CH₂).

EXAMPLE 10 3-Indan-1-ylidene-1,3-dihydroindol-2-one

A mixture of 0.4 g indan-2-one, 1.33 g oxindole and 0.5 ml piperidine in3 ml dimethylforamide was heated in a sealed tube at 130° C. for 60hours. Enough water was added to cause precipitation of an oily solid.The mixture was decanted and the remaining oily solid heated with 5 mlof ethanol using a heat gun. Upon cooling, the solid was filtered off.The solid was slurried with 3 ml of ethanol and filtered to give 160 mgof 3-indan-1-ylidene-1,3-dihydroindol-2-one as an orange solid.

¹HNMR (360 MHz d6-DMSO) δ: 10.5(s, 1H, CONH), 9.5, 7.6, 7.4, 7.4, 7.3,7.2, 7.0, 6.8 (8×m, 8H, aromatic); 3.3, 3.2 (2×m, 4H, CH₂ CH₂CH₂), 3.5(s, 3H, CH₃). MS (m/z): 248.

EXAMPLE 11[6-Methoxy-3-(2-oxo-1,2-dihydroindol-3-ylidene)-indan-1-yl]acetic Acid

A mixture of 0.66 g 5-methoxyindan-3-one acetic acid, 2.4 g oxindole and1.0 ml piperidine in 3 ml of dimethylformamide was heated in a sealedtube at 130° C. for 14 hours. Three ml of 6N hydrochloric acid was addedto the cooled reaction mixture followed by 3 ml of water. Thesupernatant was then decanted. The oily residue was heated in 5 ml ofethanol until it dissolved, the solution was then cooled and water wasadded. After standing overnight, a solid formed which was filtered andwashed with ethanol. The solid was slurried in 4 ml of ethyl acetate at70° C. for 45 minutes, filtered and suctioned dried to give 300 mg of[6-methoxy-3-(2-oxo-1,2-dihydroindol-3-ylidene)-indan-1-yl]-acetic acid.

¹HNMR (360 MHz d6-DMSO) δ: 12.3(s, 1H, COOH), 10.5(S, 1H, CONH), 9.5,7.5, 7.2, 7.1, 7.0, 6.9, 6.8 (7×m, 8H, aromatic), 3.8 (s, 3H, CH₃), 3.6,3.0 (2×m, 4H, 2×CH₂), 2.5 (m, 1H, CH). MS (m/z): 334.

EXAMPLE 12 3-(5,6-Dimethoxyindan-1-ylidene)-1,3-dihydroindol-2-one

A mixture of 0.6 g 5,6-dimethoxy-1-indanone, 1.04 g oxindole and 2.3 mlpiperidine in 4 ml dimethylformamide was heated in a sealed tube at 130°C. for 12 hours to yield an orange suspension. The mixture was added to1N hydrochloric acid in ethanol and the solids which formed werefiltered and rinsed with water and ethanol. The solid was slurried inethanol and filtered to yield 200 mg of3-(5,6-dimethoxyindan-1-ylidene)-1,3-dihydroindol-2-one.

¹HNMR (360 MHz d6-DMSO) δ: 10.3s, 1H, CONH), 9.4, 7.5, 7.2, 7.0, 6.9,6.8 (6×m, 6H, aromatic), 3.7, 3.8 (2×s, 6H, 2×CH₃), 2.8, 2.6 (2×m, 4H,CH₂CH₂). MS (m/z): 308.

EXAMPLE 13[6-Methoxy-3-(2-oxo-1,2-dihydroindol-3-ylidene)-indan-1-yl]acetic AcidSodium Salt

A suspension of 8 g[6-methoxy-3-(2-oxo-1,2-dihydroindol-3-ylidene)indan-1-yl]-acetic acidin 60 ml of water was added to 0.9 g of sodium hydroxide in 10 ml ofwater. The mixture was stirred at room temperature for 30 minutes andfiltered. The filtrate was frozen and lyophilized to give 8 g of[6-methoxy-3-(2-oxo-1,2-dihydroindol-3-ylidene)-indan-1-yl]-acetic acidsodium salt.

¹HNMR (360 MHz d6-DMSO) δ: 10.5 (S, 1H, CONH), 9.5, 7.5, 7.2, 7.1, 7.0,6.8, 6.8 (7×m, 8H, aromatic), 3.8 (s, 3H, CH₃), 3.7, 3.6, 3.0, 2.0 (4×m,4H, 2×CH₃), 2.5 (m, 1H, CH). MS (m/z): 336.

EXAMPLE 14 1′,5′,6′,7′-Tetrahydro-1H-[3,4′]biindolyliden-2-one

A mixture of 680 mg 1,5,6,7-tetrahydro-4H-indol-4-one and 1.33 goxindole in 3 ml dimethylforamide was heated at 140° C. for 50 hours.The mixture was diluted with water and extracted with ethyl acetate. Theorganic extracts were washed with water and then brine, dried oversodium sulfate, filtered and concentrated. The crude product waspurified on a silica gel column using hexanes/ethyl acetate as theeluent to provide 150 mg of1′,5′,6′,7′-tetrahydro-1H-[3,4′]biindolyliden-2-one as a yellow solid.

¹HNMR (d6-DMSO) δ: 11.1 (s, 1H, pyrrole NH), 10.2 (S, 1H, CONH), 7.9,7.0, 6.8, 6.5 (m, 6H, aromatic), 3.2, 2.8, 1.9 (3×m, 6H, 3×CH₂). MS(m/z): 251 [M+1].

6. BIOLOGICAL EVALUATION

It will be appreciated that, in any given series of compounds, aspectrum of biological activities will be obtained. In its preferredembodiments, this invention relates to novel geometrically restricted2-indolinones demonstrating the ability to modulate RTK, CTK, and STKactivity. The following assays are employed to select those compoundsdemonstrating the optimal degree of the desired activity.

Assay Procedures.

The following in vitro assays may be used to determine the level ofactivity and effect of the different compounds of the present inventionon one or more of the PKs. Similar assays can be designed along the samelines for any PK using techniques well known in the art.

The cellular/catalytic assays described herein are performed in an ELISAformat. The.general procedure is as follows: a compound is introduced tocells expressing the test kinase, either naturally or recombinantly, fora selected period of time after which, if the test kinase is a receptor,a ligand known to activate the receptor is added. The cells are lysedand the lysate is transferred to the wells of an ELISA plate previouslycoated with a specific antibody recognizing the substrate of theenzymatic phosphorylation reaction. Non-substrate components of the celllysate are washed away and the amount of phosphorylation on thesubstrate is detected with an antibody specifically recognizingphosphotyrosine compared with control cells that were not contacted witha test compound.

The cellular/biologic assays described herein measure the amount of DNAmade in response to activation of a test kinase, which is a generalmeasure of a proliferative response. The general procedure for thisassay is as follows: a compound is introduced to cells expressing thetest kinase, either naturally or recombinantly, for a selected period oftime after which, if the test kinase is a receptor, a ligand known toactivate the receptor is added. After incubation at least overnight, aDNA labeling reagent such as Bromodeoxyuridine (BrdU) or 3H-thymidine isadded. The amount of labeled DNA is detected with either an anti-BrdUantibody or by measuring radioactivity and is compared to control cellsnot contacted with a test compound.

Cellular/Catalytic Assays

Enzyme linked immunosorbent assays (ELISA) may be used to detect andmeasure the presence of PK activity. The ELISA may be conductedaccording to known protocols which are described in, for example,Voller, et al., 1980, “Enzyme-Linked Immunosorbent Assay,” In: Manual ofClinical Immunology, 2d ed., edited by Rose and Friedman, pp 359-371 Am.Soc. Of Microbiology, Washington, D.C.

The disclosed protocol may be adapted for determining activity withrespect to a specific PK. That is, the preferred protocols forconducting the ELISA experiments for specific PKs is provided below.However, adaptation of these protocols for determining a compound'sactivity for other members of the RTK family, as well as for CTKs andSTKs, is well within the scope of knowledge of those skilled in the art.

FLK-1 Assay

An ELISA assay is conducted to measure the kinase activity of the FLK-1receptor and more specifically, the inhibition or activation of TKactivity on the FLK-1 receptor. Specifically, the following assay can beconducted to measure kinase activity of the FLK-1 receptor in cellsgenetically engineered to express Flk-1.

Materials and Reagents.

a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96);

b. Cappel goat anti-rabbit IgG (catalog no. 55641);

c. PBS (Gibco Catalog No. 450-1300EB);

d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and 0.1% Tween-20);

e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4° C.);

f. HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM NaCl, 0.2% TritonX-100, and 10% glycerol);

g. EDTA (0.5 M (pH 7.0) as a 100×stock);

h. Sodium orthovanadate (0.5 M as a 100×stock);

i. Sodium pyrophosphate (0.2 M as a 100×stock);

j. NUNC 96 well V bottom polypropylene plates (Applied ScientificCatalog No. AS-72092);

k. NIH3T3 C7#3 Cells (FLK-1 expressing cells);

l. DMEM with 1×high glucose L-Glutamine (catalog No. 11965-050);

m. FBS, Gibco (catalog no. 16000-028);

n. L-glutamine, Gibco (catalog no. 25030-016);

o. VEGF, PeproTech, Inc. (catalog no. 100-20)(kept as 1 μg/100 μl stockin Milli-Q dH₂O and stored at −20° C.;

p. Affinity purified anti-FLK-1 antiserum;

q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley,et al., 1990, Cancer Research 50:1550-1558);

r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011);

s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) solution(100 mM citric acid (anhydrous), 250 mM Na₂HPO₄ (pH 4.0), 0.5 mg/ml ABTS(Sigma catalog no. A-1888)), solution should be stored in dark at 4° C.until ready for use;

t. H₂O₂ (30% solution) (Fisher catalog no. H325);

u. ABTS/ H₂O₂ (15 ml ABTS solution, 2 μl H₂O₂) prepared 5 minutes beforeuse and left at room temperature;

v. 0.2 M HCl stock in H₂O;

w. dimethylsulfoxide (100%)(Sigma Catalog No. D-8418); and

y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).

Protocol.

1. Coat Corning 96-well ELISA plates with 1.0 pg per well CappelAnti-rabbit IgG antibody in 0.1M Na₂CO₃ pH 9.6. Bring final volume to150 μl per well. Coat plates overnight at 4° C. Plates can be kept up totwo weeks when stored at 4° C.

2. Grow cells in Growth media (DMEM, supplemented with 2.0 mML-Glutamine, 10% FBS) in suitable culture dishes until confluent at 37°C., 5% CO₂.

3. Harvest cells by trypsinization and seed in Corning 25850 polystyrene96-well round bottom cell plates, 25,000 cells/well in 200 μl of growthmedia.

4. Grow cells at least one day at 37° C., 5% CO₂.

5. Wash cells with D-PBS 1×.

6. Add 200 μl/well of starvation media (DMEM, 2.0 mM 1-Glutamine, 0.1%FBS). Incubate overnight at 37° C., 5% CO₂.

7. Dilute Compounds 1:20 in polypropylene 96 well plates usingstarvation media. Dilute dimethylsulfoxide 1:20 for use in controlwells.

8. Remove starvation media from 96 well cell culture plates and add 162μl of fresh starvation media to each well.

9. Add 18 μl of 1:20 diluted compound dilution (from step 7) to eachwell plus the 1:20 dimethylsulfoxide dilution to the control wells(+/−VEGF), for a final dilution of 1:200 after cell stimulation. Finaldimethylsulfoxide is 0.5%. Incubate the plate at 37° C., 5% CO₂ for twohours.

10. Remove unbound antibody from ELISA plates by inverting plate toremove liquid. Wash 3 times with TBSW+0.5% ethanolamine, pH 7.0. Pat theplate on a paper towel to remove excess liquid and bubbles.

11. Block plates with TBSW+0.5% ethanolamine, pH 7.0, 150 μl per well.Incubate plate thirty minutes while shaking on a microtiter plateshaker.

12. Wash plate 3 times as described in step 10.

13. Add 0.5 μg/well affinity purified anti-FLU-1 polyclonal rabbitantiserum. Bring final volume to 150 μl/well with TBSW+0.5% ethanolaminepH 7.0. Incubate plate for thirty minutes while shaking.

14. Add 180 μl starvation medium to the cells and stimulate cells with20 μl/well 10.0 mM sodium orthovanadate and 500 ng/ml VEGF (resulting ina final concentration of 1.0 mM sodium orthovanadate and 50 ng/ml VEGFper well) for eight minutes at 37° C., 5% CO₂. Negative control wellsreceive only starvation medium.

15. After eight minutes, media should be removed from the cells andwashed one time with 200 μl/well PBS.

16. Lyse cells in 150 μl/well HNTG while shaking at room temperature forfive minutes. HNTG formulation includes sodium ortho vanadate, sodiumpyrophosphate and EDTA.

17. Wash ELISA plate three times as described in step 10.

18. Transfer cell lysates from the cell plate to ELISA plate andincubate while shaking for two hours. To transfer cell lysate pipette upand down while scrapping the wells.

19. Wash plate three times as described in step 10.

20. Incubate ELISA plate with 0.02 μg/well UB40 in TBSW+05%ethanolamine. Bring final volume to 150 μl/well. Incubate while shakingfor 30 minutes.

21. Wash plate three times as described in step 10.

22. Incubate ELISA plate with 1:10,000 diluted EIA grade goat anti-mouseIgG conjugated horseradish peroxidase in TBSW plus 0.5% ethanolamine, pH7.0. Bring final volume to 150 μl/well. Incubate while shaking forthirty minutes.

23. Wash plate as described in step 10.

24. Add 100 μl of ABTS/H₂O₂ solution to well. Incubate ten minutes whileshaking.

25. Add 100 μl of 0.2 M HCl for 0.1 M HCl final concentration to stopthe color development reaction. Shake 1 minute at room temperature.Remove bubbles with slow stream of air and read the ELISA plate in anELISA plate reader at 410 nm.

HER-2 ELISA

EGF Receptor-HER2 Chimeric Receptor Assay in Whole Cells.

HER2 kinase activity in whole EGFR-NIH3T3 cells are measured asdescribed below:

Materials and Reagents.

a. EGF: stock concentration: 16.5 ILM; EGF 201, TOYOBO, Co., Ltd. Japan.

b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellulardomain).

c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal)(see, Fendley,et al., supra).

d. Detection antibody: Goat anti-rabbit lgG horseradish peroxidaseconjugate, TAGO, Inc., Burlingame, Calif.

e. TBST buffer: Tris-HCl, pH 7.2 50 mM NaCl 150 mM Triton X-100 0.1 f.HNTG 5X stock: HEPES 0.1 M NaCl 0.75 M Glycerol  50% Triton X-100 1.0%g. ABTS stock: Citric Acid 100 mM Na₂HPO₄ 250 mM HCl, conc. 0.5 mM ABTS*0.5 mg/ml *(2,2‘-azinobis(3-ethylbenzthiazolinesulfonic acid)).

Keep solution in dark at 4° C. until use.

h. Stock reagents of:

EDTA 100 mM pH 7.0

Na₃VO₄ 0.5 M

Na₄ (P₂O₇) 0.2 M

Procedure.

Pre-coat ELISA Plate

1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101antibody at 0.5 μg per well in PBS, 100 μl final volume/well, and storeovernight at 4° C. Coated plates are good for up to 10 days when storedat 4° C.

2. On day of use, remove coating buffer and replace with 100 μl blockingbuffer (5% Carnation Instant Non-Fat Dry Milk in PBS). Incubate theplate, shaking, at room temperature (about 23° C. to 25° C.) for 30minutes. Just prior to use, remove blocking buffer and wash plate 4times with TBST buffer.

Seeding Cells

1. An NIH3T3 cell line overexpressing a chimeric receptor containing theEGFR extracellular domain and intracellular HER2 kinase domain can beused for this assay.

2. Choose dishes having 80-90% confluence for the experiment. Trypsinizecells and stop reaction by adding 10% fetal bovine serum. Suspend cellsin DMEM medium (10% CS DMEM medium) and centrifuge once at 1500 rpm, atroom temperature for 5 minutes.

3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), andcount the cells using trypan blue. Viability above 90% is acceptable.Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000cells per well, 100 μl per well, in a 96 well microtiter plate. Incubateseeded cells in 5% CO₂ at 37° C. for about 40 hours.

Assay Procedures

1. Check seeded cells for contamination using an inverted microscope.Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer5 μl to a TBST well for a final drug dilution of 1:200 and a final DMSOconcentration of 1%. Control wells receive DMSO alone. Incubate in 5%CO₂ at 37° C. for two hours.

2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of10 μl dilute EGF (1:12 dilution), 100 nM final concentration isattained.

3. Prepare fresh HNTG* sufficient for 100 μl per well; and place on ice.

HNTG* (10 ml): HNTG stock 2.0 ml milli-Q H₂O 7.3 ml EDTA, 100 mM, pH 7.00.5 ml Na₃VO₄ (0.5 M) 0.1 ml Na₄(P₂O₇) (0.2 M) 0.1 ml

4. After 120 minutes incubation with drug, add prepared SGF ligandto.cells, 10 μl per well, to a final concentration of 100 nM. Controlwells receive DMEM alone. Incubate with shaking, at room temperature,for 5 minutes.

5. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG*to cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, removeblocking buffer from other ELISA plate and wash with TBST as describedabove.

6. With a pipette tip securely fitted to a micropipettor, scrape cellsfrom plate and homogenize cell material by repeatedly aspirating anddispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked,and washed ELISA plate. Incubate shaking at room temperature for onehour.

7. Remove lysate and wash 4 times with TBST. Transfer freshly dilutedanti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shakingat room temperature for 30 minutes in the presence of the anti-Ptyrantiserum (1:3000 dilution in TBST).

8. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transferthe freshly diluted TAGO anti-rabbit IgG antibody to the ELISA plate at100 μl per well. Incubate shaking at room temperature for 30 minutes(anti-rabbit IgG antibody: 1:3000, dilution in TBST).

9. Remove TAGO detection antibody and wash 4 times with TBST. Transferfreshly prepared ABTS/H₂O₂ solution to ELISA plate, 100 μl per well.Incubate shaking at room temperature for 20 minutes. (ABTS/H₂O₂solution: 1.0 μl 30% H₂O₂ in 10 ml ABTS stock).

10. Stop reaction by adding 50 μl 5N H₂SO₄ (optional), and determineO.D. at 410 nm.

11. The maximal phosphotyrosine signal is determined by subtracting thevalue of the negative controls from the positive controls. The percentinhibition of phosphotyrosine content for extract-containing wells isthen calculated, after subtraction of the negative controls.

PDGF-R Assay

All cell culture media, glutamine, and fetal bovine serum can bepurchased from Gibco Life Technologies (Grand Island, N.Y.) unlessotherwise specified. All cells are grown in a humid atmosphere of 90-95%air and 5-10% CO₂ at 37° C. All cell lines are routinely subculturedtwice a week and are negative for mycoplasma as determined by theMycotect method (Gibco).

For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU)are grown to 80-90% confluency in growth medium (MEM with 10% FBS, NEAA,1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in0.5% serum at 25,000 to 30,000 cells per well. After overnightincubation in 0.5% serum-containing medium, cells are changed toserum-free medium and treated with test compound for 2 hr in a 5% CO₂,37° C. incubator. Cells are then stimulated with ligand for 5-10 minutefollowed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5mM EDTA, 5 mM Na₃VO₄, 0.2% Triton X-100, and 2 mM NaPyr). Cell lysates(0.5 mg/well in PBS) are transferred to ELISA plates previously coatedwith receptor-specific antibody and which had been blocked with 5% milkin TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X-100) atroom temperature for 30 min. Lysates are incubated with shaking for 1hour at room temperature. The plates are washed with TBST four times andthen incubated with polyclonal anti-phosphotyrosine antibody at roomtemperature for 30 minutes. Excess anti-phosphotyrosine antibody isremoved by rinsing the plate with TBST four times. Goat anti-rabbit IgGantibody is added to the ELISA plate for 30 min at room temperaturefollowed by rinsing with TBST four more times. ABTS (100 mM citric acid,250 mM Na₂HPO₄ and 0.5 mg/mL2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H₂O₂ (1.2 mL30% H₂O₂ to 10 ml ABTS) is added to the ELISA plates to start colordevelopment. Absorbance at 410 nm with a reference wavelength of 630 nmis recorded about 15 to 30 min after ABTS addition.

IGF-1 RECEPTOR

The following protocol may be used to measure phosphotyrosine level onIGF-1 receptor, which indicates IGF-1 receptor tyrosine kinase activity.

Materials and Reagents.

a. The cell line used in this assay is 3T3/IGF-1R, a cell linegenetically engineered to overexpresses IGF-1 receptor.

b. NIH3T3/IGF-1R is grown in an incubator with 5% CO₂ at 37° C. Thegrowth media is DMEM+10% FBS (heat inactivated)+2 mM L-glutamine.

c. Affinity purified anti-IFG-1R antibody 17-69.

d. D-PBS: KH₂PO₄ 0.20 g/l KH₂PO₄ 2.16 g/l KCl 0.20 g/l NaCl 8.00 g/l (pH7.2) e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-FatDry Milk). f. TBST buffer: Tris-HCl   50 mM NaCl  150 mM (pH 7.2/HCl10N) Triton X-100 0.1% Stock solution of TBS (10X) is prepared, andTriton X-100 is added to the buffer during dilution. g HNTG buffer:HEPES   20 mM NaCl  150 mM (pH 7.2/HCl 1N) Glycerol 10% Triton X-1000.2% Stock solution (5X) is prepared and kept at 4° C.

h. EDTA/HCl: 0.5 M pH 7.0 (NaOH) as 100×stock.

i. Na₃VO₄: 0.5 M as 100×stock and aliquots are kept at 80° C.

j. Na₄P₂O₇: 0.2 M as 100×stock.

k. Insulin-like growth factor-1 from Promega (Cat#G5111).

l. Rabbit polyclonal anti-phosphotyrosine antiserum.

m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (Cat.No. 4520, Lot No. 1802): Tago, Inc., Burlingame, Calif.

n. ABTS (2,2′-azinobis (3-ethylbenzthiazolinesulfonic acid)) solution:

Citric acid 100 mM Na₂HPO₄ 250 mM (pH 4.0/1 N HCl) ABTS  0.5 mg/ml

ABTS solution should be kept in dark and 4° C. The solution should bediscarded when it turns green.

o. Hydrogen Peroxide: 30% solution is kept in the dark and at 4° C.

Procedure.

All the following steps are conducted at room temperature unlessspecifically indicated otherwise. All ELISA plate washings are performedby rinsing the plate with tap water three times, followed by one TBSTrinse. Pat plate dry with paper towels.

Cell Seeding:

1. The cells, grown in tissue culture dish (Corning 25020-100) to 80-90%confluence, are harvested with Trypsin-EDTA (0.25%, 0.5 ml/D-100,GIBCO).

2. Resuspend the cells in fresh DMEM+10% FBS+2 mM L-Glutamine, andtransfer to 96-well tissue culture plate (Corning, 25806-96) at 20,000cells/well (100 μl/well). Incubate for 1 day then replace medium toserum-free medium (90/μl) and incubate in 5% CO₂ and 37° C. overnight.

ELISA Plate Coating and Blocking:

1. Coat the ELISA plate (Corning 25805-96) with Anti-IGF-1R Antibody at0.5 μg/well in 100 μl PBS at least 2 hours.

2. Remove the coating solution, and replace with 100 μl Blocking Buffer,and shake for 30 minutes. Remove the blocking buffer and wash the platejust before adding lysate.

Assay Procedures:

1. The drugs are tested under serum-free condition.

2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-wellpoly-propylene plate, and transfer 10 μl/well of this solution to thecells to achieve final drug dilution 1:100, and final DMSO concentrationof 1.0%. Incubate the cells in 5% CO₂ at 37° C. for 2 hours.

3. Prepare fresh cell lysis buffer (HNTG*)

HNTG   2 ml EDTA 0.1 ml Na₃VO₄ 0.1 ml Na₄(P₂O₇) 0.1 ml H₂O 7.3 ml

4. After drug incubation for two hours, transfer 10 μl/well of 200 nMIGF-1 Ligand in PBS to the cells (Final Conc. is 20 nM), and incubate at5% CO₂ at 37° C. for 10 minutes.

5. Remove media and add 100 μl/well HNTG* and shake for 10 minutes. Lookat cells under microscope to see if they are adequately lysed.

6. Use a 12-channel pipette to scrape the cells from the plate, andhomogenize the lysate by repeated aspiration and dispensing. Transferall the lysate to the antibody coated ELISA plate, and shake for 1 hour.

7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000 withTBST) 100 μl/well, and shake for 30 minutes.

8. Remove anti-pTyr, wash the plate, transfer TAGO (1:3,000 with TBST)100 μl/well, and shake for 30 minutes.

9. Remove detection antibody, wash the plate, and transfer freshABTS/H₂O₂ (1.2 μl H₂O₂ to 10 ml ABTS) 100 μl/well to the plate to startcolor development.

10. Measure OD at 410 nm with a reference wavelength of 630 nm inDynatec MR5000.

EGFR Assay

EGF Receptor kinase activity in cells genetically engineered to expresshuman EGF-R can be measured as described below:

Materials and Reagents.

a. EGF Ligand: stock concentration=16.5 μM; EGF 201, TOYOBO, Co., Ltd.Japan.

b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellulardomain).

c. Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).

d. Detection antibody: Goat anti-rabbit lgG horse radish peroxidaseconjugate, TAGO, Inc., Burlingame, Calif.

e. TBST buffer: Tris-HCl, pH 7 50 mM NaCl 150 mM Triton X-100 0.1 f.HNTG 5X stock: HEPES 0.1 M NaCl 0.75 M Glycerol 50 Triton X-100 1.0% g.ABTS stock: Citric Acid 100 mM Na₃VO₄ 250 mM HCl, conc. 4.0 pH ABTS* 0.5mg/ml

Keep solution in dark at 4° C. until used.

h. Stock reagents of:

EDTA 100 mM pH 7.0

Na₃VO₄ 0.5 M

Na₄(P₂O₇) 0.2 M

Procedure.

Pre-coat ELISA Plate

1. Coat ELISA plates (Corning, 96-well, Cat. #25805-96) with 05-101antibody at 0.5 μg per well in PBS, 150 μl final volume/well, and storeovernight at 4° C. Coated plates are good for up to 10 days when storedat 4° C.

2. On day of use, remove coating buffer and replace with blocking buffer(5% Carnation Instant NonFat Dry Milk in PBS). Incubate the plate,shaking, at room temperature (about 23° C. to 25° C.) for 30 minutes.Just prior to use, remove blocking buffer and wash plate 4 times withTBST buffer.

Seeding Cells

1. NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987) can beuse for this assay.

2. Choose dishes having 80-90% confluence for the experiment. Trypsinizecells and stop reaction by adding 10% CS DMEM medium. Suspend cells inDMEM medium (10% CS DMEM medium) and centrifuge once at 1000 rpm at roomtemperature for 5 minutes.

3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), andcount the cells using trypan blue. Viability above 90% is acceptable.Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000cells per well, 100 μl per well, in a 96 well microtiter plate. Incubateseeded cells in 5% CO₂ at 37° C. for about 40 hours.

Assay Procedures.

1. Check seeded cells for contamination using an inverted microscope.Dilute test compounds stock (10 mg/ml in DMSO) 1:10 in DMEM medium, thentransfer 5 μl to a test well for a test compounds drug dilution of 1:200and a final DMSO concentration of 1%. Control wells receive DMSO alone.Incubate in 5% CO₂ at 37° C. for one hour.

2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of10 μl dilute EGF (1:12 dilution), 25 nM final concentration is attained.

3. Prepare fresh 10 ml HNTG* sufficient for 100 μl per well whereinHNTG* comprises: HNTG stock (2.0 ml), milli-Q H₂O (7.3 ml), EDTA, 100mM, pH 7.0 (0.5 ml), Na₃VO₄ 0.5 M (0.1 ml) and Na₄(P₂O₇), 0.2 M (0.1ml).

4. Place on ice.

5. After two hours incubation with drug, add prepared EGF ligand tocells, 10 μl per well, to yield a final concentration of 25 nM. Controlwells receive DMEM alone. Incubate, shaking, at room temperature, for 5minutes.

6. Remove test compound, EGF, and DMEM. Wash cells twice with PBS.Transfer HNTG* to cells, 100 μl per well. Place on ice for 5 minutes.Meanwhile, remove blocking buffer from other ELISA plate and wash withTBST as described above.

7. With a pipette tip securely fitted to a micropipettor, scrape cellsfrom plate and homogenize cell material by repeatedly aspirating anddispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked,and washed ELISA plate. Incubate shaking at room temperature for onehour.

8. Remove lysate and wash 4 times with TBST. Transfer freshly dilutedanti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shakingat room temperature for 30 minutes in the presence of the anti-Ptyrantiserum (1:3000 dilution in TBST).

9. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transferthe freshly diluted TAGO 30 anti-rabbit IgG antibody to the ELISA plateat 100 μl per well. Incubate shaking at room temperature for 30 minutes(anti-rabbit IgG antibody: 1:3000 dilution in TBST).

10. Remove detection antibody and wash 4 times with TBST. Transferfreshly prepared ABTS/H₂O₂ solution to ELISA plate, 100 μl per well.Incubate at room temperature for 20 minutes. ABTS/H₂O₂ solution: 1.2 μl30% H₂O₂ in 10 ml ABTS stock.

11. Stop reaction by adding 50 μl 5N H₂SO₄ (optional), and determineO.D. at 410 nm.

12. The maximal phosphotyrosine signal is determined by subtracting thevalue of the negative controls from the positive controls. The percentinhibition of phosphotyrosine content for extract-containing wells isthen calculated, after subtraction of the negative controls.

Met Autophosphorylation Assay

This assay determines Met tyrosine kinase activity by analyzing Metprotein tyrosine kinase levels on the Met receptor. Reagents

a. HNTG (5×stock solution): Dissolve 23.83 g HEPES and 43.83 g NaCl inabout 350 ml dH₂O. Adjust pH to 7.2 with HCl or NaOH, add 500 mlglycerol and 10 ml Triton X-100, mix, add dH₂O to 1 L total volume. Tomake 1 L of 1×working solution add 200 ml 5×stock solution to 800 mldH₂O, check and adjust pH as necessary, store at 4° C.

b. PBS (Dulbecco's Phosphate-Buffered Saline), Gibco Cat. #450-130OEB(1×solution).

c. Blocking Buffer: in 500 ml dH₂O place 100 g BSA, 12.1 g Tris-pH7.5,58.44 g NaCl and 10 ml Tween-20, dilute to 1 L total volume.

d. Kinase Buffer: To 500 ml dH₂O add 12.1 g TRIS (pH 7.2), 58.4 g NaCl,40.7 g MgCl₂ and 1.9 g EGTA; bring to 1 L total volume with dH₂O.

e. PMSF (Phenylmethylsulfonyl fluoride), Sigma Cat. 190 P-7626, to 435.5mg, add 100% ethanol to 25 ml total volume, vortex.

f. ATP (Bacterial Source), Sigma Cat. #A-7699, store powder at −20° C.;to make up solution for use, dissolve 3.31 mg in 1 ml dH₂O.

g. RC-20H HRPO Conjugated Anti-Phosphotyrosine, TransductionLaboratories Cat. #E120H.

h. Pierce 1-Step™ Turbo TMB-ELISA (3,3′,5,5′-tetramethylbenzidine,Pierce Cat. #34022.

i. H₂SO₄, add 1 ml conc.(18 N) to 35 ml dH₂O.

j. TRIS HCL, Fischer Cat. #BP152-5; to 121.14 g of material, add 600 mlMilliQ H₂O, adjust pH to 7.5 (or 7.2) with HCl, bring volume to 1 L withMilliQ H₂O.

k. NaCl, Fischer Cat. #S271-10, make up 5M solution.

l. Tween-20, Fischer Cat. #S337-500.

m. Na₃VO₄, Fischer Cat. #S454-50, to 1.8 g material add 80 ml MilliQH₂O, adjust pH to 10.0 with HCl or NaOH, boil in microwave, cool, checkpH, repeat procedure until pH stable at 10.0, add MilliQ H₂O to 100 mltotal volume, make 1 ml aliquots and store at −80° C.

n. MgCl₂, Fischer Cat. #M33-500, make up 1M solution.

o. HEPES, Fischer Cat. #BP310-500, to 200 ml MilliQ H₂O, add 59.6 gmaterial, adjust pH to 7.5, bring volume to 250 ml total, sterilefilter.

p. Albumin, Bovine (BSA), Sigma Cat. #A-4503, to 30 grams material addsterile distilled water to make total volume of 300 ml, store at 4° C.

q. TBST Buffer: to approx. 900 ml dH₂O in a 1 L graduated cylinder add6.057 g TRIS and 8.766 g NaCl, when dissolved, adjust pH to 7.2 withHCl, add 1.0 ml Triton X-100 and bring to 1 L total volume with dH₂O.

r. Goat Affinity purified antibody Rabbit IgG (whole molecule), CappelCat. #55641.

s. Anti h-Met (C-28) rabbit polyclonal IgG antibody, Santa Cruz ChemicalCat. #SC-161.

t. Transiently Transfected EGFR/Met chimeric cells (EMR) (Komada, etal., Oncogene, 8:2381-2390 (1993).

u. Sodium Carbonate Buffer, (Na₂CO₄, Fischer Cat. #S495): to 10.6 gmaterial add 800 ml MilliQ H₂O, when dissolved adjust pH to 9.6 withNaOH, bring up to 1 L total volume with MilliQ H₂O, filter, store at 4°C.

Procedure

All of the following steps are conducted at room temperature unless itis specifically indicated otherwise. All ELISA plate washing is byrinsing 4× with TBST.

EMR Lysis

This procedure can be performed the night before or immediately prior tothe start of receptor capture.

1. Quick thaw lysates in a 37° C. waterbath with a swirling motion untilthe last crystals disappear.

2. Lyse cell pellet with 1×HNTG containing 1 mM PMSF. Use 3 ml of HNTGper 15 cm dish of cells. Add 1/2 the calculated HNTG volume, vortex thetube for 1 min., add the remaining amount of HNTG, vortex for anothermin.

3. Balance tubes, centrifuge at 10,000× g for 10 min at 4° C.

4. Pool supernatants, remove an aliquot for protein determination.

5. Quick freeze pooled sample in dry ice/ethanol bath. This step isperformed regardless of whether lysate will be stored overnight or usedimmediately following protein determination.

6. Perform protein determination using standard bicinchoninic acid (BCA)method (BCA Assay Reagent Kit from Pierce Chemical Cat. #23225).

ELISA Procedure

1. Coat Corning 96 well ELISA plates with 5 μg per well Goat anti-Rabbitantibody in Carbonate Buffer for a total well volume of 50 μl. Storeovernight at 4° C.

2. Remove unbound Goat anti-rabbit antibody by inverting plate to removeliquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. withshaking.

4. Wash 4× with TBST. Pat plate on a paper towel to remove excess liquidand bubbles.

5. Add 1 μg per well of Rabbit anti-Met antibody diluted in TBST for atotal well volume of 100 μl.

6. Dilute lysate in HNTG (90 μg lysate/100 μl)

7. Add 100 μl of diluted lysate to each well. Shake for 60 min.

8. Wash 4× with TBST. Pat on paper towel to remove excess liquid andbubbles.

9. Add 50 μl of 1×lysate buffer per well.

10. Dilute compounds/extracts 1:10 in 1×Kinase Buffer in a polypropylene96 well plate.

11. Transfer 5.5 μl of diluted compound to ELISA plate wells. Incubateat room temperature with shaking for 20 min.

12. Add 5.5 μl of 60 μM ATP solution per well. Negative controls do notreceive any ATP. Incubate for 90 min., with shaking.

13. Wash 4× with TBST. Pat plate on paper towel to remove excess liquidand bubbles.

14. Add 100 μl per well of RC20 (1:3000 dilution in Blocking Buffer).Incubate 30 min. with shaking.

15. Wash 4× with TBST. Pat plate on paper towel to remove excess liquidand bubbles.

16. Add 100 μl per well of Turbo-TMB. Incubate with shaking for 30-60min.

17. Add 100 μl per well of 1M H₂SO₄ to stop reaction.

18. Read assay on Dynatech MR7000 ELISA reader. Test Filter=450 nm,reference filter=410 nm.

Biochemical src Assay

This assay is used to determine src protein kinase activity measuringphosphorylation of a biotinylated peptide as the readout.

Materials and Reagents:

a. Yeast transformed with src (Sugen, Inc., Redwood City, Calif.).

b. Cell lysates: Yeast cells expressing src are pelleted, washed oncewith water, re-pelleted and stored at −80° C. until use.

c. N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is prepared by standardprocedures well known to those skilled in the art.

d. DMSO: Sigma, St. Louis, Mo.

e. 96 Well ELISA Plate: Corning 96 Well Easy Wash, Modified flat BottomPlate, Corning Cat. #25805-96.

f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds:Applied Scientific Cat. #A-72092.

g. Vecastain ELITE ABC reagent: Vector, Burlingame, Calif.

h. Anti-src (327) mab: Schizosaccharomyces Pombe is used to expressrecombinant Src (Superti-Furga, et al., EMBO J., 12:2625-2634;Superti-Furga, et al., Nature Biochem., 14:600-605). S. Pombe strainSP200 (h-s leul.32 ura4 ade2110) is grown as described andtransformations are pRSP expression plasmids are done by the lithiumacetate method (Superti-Furga, supra). Cells are grown in the presenceof 1 μM thiamine to repress expression from the nmtl promoter or in theabsence of thiamine to induce expression.

i. Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40 may be usedinstead).

j. Turbo TMB-ELISA peroxidase substrate: Pierce Chemical.

Buffer Solutions

a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO PBS, GIBCO-Cat.#450-1300EB.

b. Blocking Buffer: 5% Non-fat milk (Carnation) in PBS.

c. Carbonate Buffer: Na₂CO₄ from Fischer, Cat. #S495, make up 100 mMstock solution.

d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl₂; 0.2 ml (from a1M stock solution) MnCl₂; 0.2 ml (from a 1M stock solution) DTT; 5.0 ml(from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 ml totalvolume with MilliQ H₂O.

e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from5M-stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1ml Na₃VO₄ (from a 0.1 M stock solution); bring to 100 ml total volumewith MilliQ H₂O.

f. ATP: Sigma Cat. #A-7699, make up 10 mM stock solution (5.51 mg/ml).

g. TRIS-HCl: Fischer Cat. #BP 152-5, to 600 ml MilliQ H₂O add 121.14 gmaterial, adjust pH to 7.5 with HCl, bring to 1 L total volume withMilliQ H₂O.

h. NaCl: Fischer Cat. #S271-10, Make up 5M stock solution with MilliQH₂O.

NaVO₄: Fischer Cat. #S454-50; to 80 ml MilliQ H₂O, add 1.8 g material;adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool; check pH,repeat pH adjustment until pH remains stable after heating/coolingcycle; bring to 100 ml total volume with MilliQ H₂O; make 1 ml aliquotsand store at −80° C.

j. MgCl₂: Fischer Cat. #M33-500, make up 1M stock solution with MilliQH₂O.

k. HEPES: Fischer Cat. #BP 310-500; to 200 ml MilliQ H₂O, add 59.6 gmaterial, adjust pH to 7.5, bring to 250 ml total volume with MilliQH₂O, sterile filter (1M stock solution).

l. TBST Buffer: TBST Buffer: To 900 ml dH₂O add 6.057 g TRIS and 8.766 gNaCl; adjust pH to 7.2 with HCl, add 1.0 ml. Triton-X100; bring to 1 Ltotal volume with dH₂O.

m. MnCl₂: Fischer Cat. #M87-100, make up 1M stock solution with MilliQH₂O.

n. DTT: Fischer Cat. #BP172-5.

o. TBS (TRIS Buffered Saline): to 900 ml MilliQ H₂O add 6.057 g TRIS and8.777 g NaCl; bring to 1 L total volume with MilliQ H₂O.

p. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 mlKinase Buffer, 200 μg GST-ζ, bring to final volume of 8.0 ml with MilliQH₂O.

q. Biotin labeled EEEYEEYEEEYEEEYEEEY: Make peptide stock solution (1mM, 2.98 mg/ml) in water fresh just before use.

r. Vectastain ELITE ABC reagent: To prepare 14 ml of working reagent,add 1 drop of reagent A to 15 ml TBST and invert tube several times tomix. Then add 1 drop of reagent B. Put tube on orbital shaker at roomtemperature and mix for 30 minutes.

Procedures:

Preparation of src Coated ELISA Plate.

1. Coat ELISA plate with 0.5 μg/well anti-src mab in 100 μl of pH 9.6sodium carbonate buffer; hold at 4° C. overnight.

2. Wash wells once with PBS.

3. Block plate with 0.15 ml 5% milk in PBS for 30 min. at roomtemperature.

4. Wash plate 5× with PBS.

5. Add 10 μg/well of src transformed yeast lysates diluted in LysisBuffer (0.1 ml total volume per well). (Amount of lysate may varybetween batches.) Shake plate for 20 minutes at room temperature.

Preparation of Phosphotyrosine Antibody-coated ELISA Plate.

1. 4G10 plate: coat 0.5 μg/well 4G10 in 100 μl PBS overnight at 4° C.and block with 150 μl of 5% milk in PBS for 30 minutes at roomtemperature.

Kinase Assay Procedure.

1. Remove unbound proteins.from plates and wash plates 5× with PBS.

2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 μl of10×-Kinase Buffer and 10 μM (final concentration)biotin-EEEYEEYEEEYEEEYEEEY per well diluted in water.

3. Add 10 μl of compound diluted in water containing 10% DMSO andpre-incubate for 15 minutes at room temperature.

4. Start kinase reaction by adding 10 μl/well of 0.05 mM ATP in water (5μM ATP final).

5. Shake ELISA plate for 15 min. at room temperature.

6. Stop kinase reaction by adding 10 μl of 0.5 M EDTA per well.

7. Transfer 90 μl supernatant to a blocked 4G10 coated ELISA plate.

8. Incubate for 30 min. while shaking at room temperature.

9. Wash plate 5× with TBST.

10. Incubate with Vectastain ELITE ABC reagent (100 μl/well) for 30 min.at room temperature.

11. Wash the wells 5× with TBST.

12. Develop with Turbo TMB.

Biochemical lck Assay

This assay is used to determine lck protein kinase activities measuringphosphorylation of GST-ζ as the readout.

Materials and Reagents:

Yeast transformed with lck. Schizosaccharomyces Pombe is used to expressrecombinant Lck (Superti-Furga, et al., EMB) J, 12:2625-2634;Superti-Furga, et al., Nature Biotech., 14:600-605). S. Pombe strainSP200 (h-s leul.32 ura4 ade210) is grown as described andtransformations with pRSP expression plasmids are done by the lithiumacetate method (Superti-Furga, supra). Cells are grown in the presenceof 1 μM thiamine to induce expression.

b. Cell lysates: Yeast cells expressing lck are pelleted, washed once inwater, re-pelleted and stored-frozen at −80° C. until use.

c. GST-ζ: DNA encoding for GST-ζ fusion protein for expression inbacteria obtained from Arthur Weiss of the Howard Hughes MedicalInstitute at the University of California, San Francisco. Transformedbacteria are grown overnight while shaking at 25° C. GST-ζ is purifiedby glutathione affinity chromatography, Pharmacia, Alameda, Calif.

d. DMSO: Sigma, St. Louis, Mo.

e. 96-Well ELISA plate: Corning 96 Well Easy Wash, Modified Flat BottomPlate, Corning Cat. #25805-96.

f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds:Applied Scientific Cat. #AS-72092.

g. Purified Rabbit anti-GST antiserum: Amrad Corporation (Australia)Cat. #90001605.

h. Goat anti-Rabbit-IgG-HRP: Amersham Cat. #V010301.

i. Sheep ant-mouse IgG (H+L): Jackson Labs Cat. #5215-005-003.

j. Anti-Lck (3A5) mab: Santa Cruz Biotechnology Cat #sc-433.

k. Monoclonal anti-phosphotyrosine UBI 05-321 (UB40 may be usedinstead).

Buffer Solutions:

a. PBS (Dulbecco's Phosphate-Buffered Saline) 1×solution: GIBCO PBS,GIBCO Cat. #450-1300EB.

b. Blocking. Buffer: 100 g. BSA, 12.1 g. TRIS (pH7.5), 58.44 g NaCl, 10ml Tween-20, bring up to 1 L total volume with MilliQ H₂O.

c. Carbonate Buffer: Na₂CO₄ from Fischer, Cat. #S495; make up 100 mMsolution with MilliQ H₂O.

d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl₂; 0.2 ml (from a1M stock solution) MnCl₂; 0.2 ml (from a 1M stock solution) DTT;. 5.0 ml(from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 ml totalvolume with MilliQ H₂O.

e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1ml Na₃VO₄ (from a 0.1 M stock solution); bring to 100 ml total volumewith MilliQ H₂O.

f. ATP: Sigma Cat. #A-7699, make up 10 mM stock solution (5.51 mg/ml).

g. TRIS-HCl: Fischer Cat. #BP 152-5, to 600 ml MilliQ H₂O add 121.14 gmaterial, adjust pH to 7.5 with HCl, bring to 1 L total volume withMilliQ H₂O.

h. NaCl: Fischer Cat. #S271-10, Make up 5M stock solution with MilliQH₂O.

i Na₃VO₄: Fischer Cat. #S454-50; to 80 ml MilliQ H₂O, add 1.8 gmaterial; adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool;check pH, repeat pH adjustment until pH remains stable afterheating/cooling cycle; bring to 100 ml total volume with MilliQ H₂O;make 1 ml aliquots and store at −80° C.

j. MgCl₂: Fischer Cat. #M33-7500, make up 1M stock solution with MilliQH₂O.

k. HEPES: Fischer Cat. #BP 310-500; to 200 ml MilliQ H₂O, add 59.6 gmaterial, adjust pH to 7.5, bring to 250 ml total volume with MilliQH₂O, sterile filter (1M stock solution).

l. Albumin, Bovine (BSA), Sigma Cat. #A4503; to 150 ml MilliQ H₂O add 30g material, bring 300 ml total volume with MilliQ H₂O, filter through0.22 μm filter, store at 4° C.

m. TBST Buffer: To 900 ml dH₂O add 6.057 g TRIS and 8.766 g NaCl; adjustpH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to 1 L total volumewith dH₂O.

n. MnCl₂: Fischer Cat. #M87-100, make up 1M stock solution with MilliQH₂O.

o. DTT; Fischer Cat. #BP172-5.

p. TBS (TRIS Buffered Saline): to 900 ml MilliQ H₂O add 6.057 g TRIS and8.777 g NaCl; bring to 1 L total volume with MilliQ H₂O.

q. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 mlKinase Buffer, 200 μg GST-ζ, bring to final volume of 8.0 ml with MilliQH₂O.

Procedures:

Preparation of Lck Coated ELISA Plate.

1. Coat 2.0 μg/well Sheep anti-mouse IgG in 100 μl of pH 9.6 sodiumcarbonate buffer at 4° C. overnight.

2. Wash well once with PBS.

3. Block plate with 0.15 ml of blocking Buffer for 30 min. at room temp.

4. Wash plate 5× with PBS.

5. Add 0.5 μg/well of anti-lck (mab 3A5) in 0.1 ml PBS at roomtemperature for 1-2 hours.

6. Wash plate 5× with PBS.

7. Add 20 μg/well of lck transformed yeast lysates diluted in LysisBuffer (0.1 ml total volume per well). Shake plate at 4° C. overnight toprevent loss of activity.

Preparation of Phosphotyrosine Antibody-coated ELISA Plate.

1. UB40 plate: 1.0 μg/well UB40 in 100 μl of PBS overnight at 4° C. andblock with 150 μl of Blocking Buffer for at least 1 hour.

Kinase Assay Procedure.

1. Remove unbound proteins from plates and wash plates 5× with PBS.

2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 μl of10×Kinase Buffer and 2 μg GST-ζ per well diluted with water).

3. Add 10 μl of compound diluted in water containing 10% DMSO andpre-incubate for 15 minutes at room temperature.

4. Start kinase reaction by adding 10μl/well of 0.1 mM ATP in water (10μM ATP final).

5. Shake ELISA plate for 60 min. at room temperature.

6. Stop kinase reaction by adding 10 μl of 0.5 M EDTA per well.

7. Transfer 90 μl supernatant to a blocked 4G10 coated ELISA plate fromsection B, above.

8. Incubate while shaking for 30 min. at room temperature.

9. Wash plate 5× with TBST.

10. Incubate with Rabbit anti-GST antibody at 1:5000 dilution in 100 μlTBST for 30 min. at room temperature.

11. Wash the wells 5× with TBST.

12. Incubate with Goat anti-Rabbit-IgG-HRP at 1:20,000 dilution in 100μl of TBST for 30 min. at room temperature.

13. Wash the wells 5× with TBST.

14. Develop with Turbo TMB.

Assay Measuring Phosphorylating Function of RAF.

The following assay reports the amount of RAF-catalyzed phosphorylationof its target protein MEK as well as MEK's target MAPK. The RAF genesequence is described in Bonner et al., 1985, Molec. Cell. Biol.,5:1400-1407, and is readily accessible in multiple gene sequence databanks. Construction of the nucleic acid vector and cell lines utilizedfor this portion of the invention are fully described in Morrison etal., 1988, Proc. Natl. Acad. Sci. USA, 85:8855-8859.

Materials and Reagents

1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL, Gaithersburg, Md.

2. RIPA buffer: 20 mM Tris/HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1 mMPMSF, 5 mg/L Aprotenin, 0.5% Triton X-100;

3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression andpurification by affinity chromatography are performed according to themanufacturer's procedures. Catalog#K 350-01 and R 350-40, InvitrogenCorp., San Diego, Calif.

4. His-MAPK (ERK 2); His-tagged MAPK is expressed in XL1 Blue cellstransformed with pUC18 vector encoding His-MAPK. His-MAPK is purified byNi-affinity chromatography. Cat#27-4949-01, Pharmacia, Alameda, Calif.,as described herein.

5. Sheep anti mouse IgG: Jackson laboratories, West Grove, Pa. Catalog,#515-006-008, Lot#28563

6. RAF-1 protein kinase specific antibody: URP2653 from UBI.

7. Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL,Gaithersburg, Md.

8. Wash buffer: TBST (50 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1% TritonX-100).

9. Block buffer: TBST, 0.1% ethanolamine pH 7.4

10. DMSO, Sigma, St. Louis, Mo.

11. Kinase buffer (KB): 20 mM HEPES/HCl pH 7.2, 150 mM NaCl, 0.1% TritonX-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 mM sodium orthovanadate, 0.5 MMDTT and 10 mM MgCl₂.

12. ATP mix: 100 mM MgCl₂, 300 mM ATP, 10 mCi γ³³P ATP (Dupont-NEN)/mL.

13. Stop solution: 1% phosphoric acid; Fisher, Pittsburgh, Pa.

14. Wallac Cellulose Phosphate Filter mats; Wallac, Turku, Finnland.

15. Filter wash solution: 1% phosphoric acid, Fisher, Pittsburgh, Pa.

16. Tomtec plate harvester, Wallac, Turku, Finnland.

17. Wallac beta plate reader #1205, Wallac, Turku, Finnland.

18. NUNC 96-well V bottom polypropylene plates for compounds AppliedScientific Catalog #AS-72092.

Procedure

All of the following steps are conducted at room temperature unlessspecifically indicated otherwise.

1. ELISA plate coating: ELISA wells are coated with 100 ml of Sheep antimouse affinity purified antiserum (1 mg/100 mL coating buffer) overnight at 4° C. ELISA plates can be used for two weeks when stored at 4°C.

2. Invert the plate and remove liquid. Add 100 mL of blocking solutionand incubate for 30 min.

3. Remove blocking solution and wash four times with wash buffer. Patthe plate on a paper towel to remove excess liquid.

4. Add 1 mg of antibody specific for RAF-1 to each well and incubate for1 hour. Wash as described in step 3.

5. Thaw lysates from RAS/RAF infected Sf9 cells and dilute with TBST to10 mg/100 mL. Add 10 mg of diluted lysate to the wells and incubate for1 hour. Shake the plate during incubation. Negative controls receive nolysate. Lysates from RAS/RAF infected Sf9 insect cells are preparedafter cells are infected with recombinant baculoviruses at a MOI of 5for each virus, and harvested 48 hours later. The cells are washed oncewith PBS and lysed in RIPA buffer. Insoluble material is removed bycentrifugation (5 min at 10,000× g). Aliquots of lysates are frozen indry ice/ethanol and stored at −80° C. until use.

6. Remove non-bound material and wash as outlined above (step 3).

7. Add 2 mg of T-MEK and 2 mg of His-MAEPK per well and adjust thevolume to 40 ml with kinase buffer. Methods for purifying T-MEK and MAPKfrom cell extracts are provided herein by example.

8. Pre-dilute compounds (stock solution 10 mg/ml DMSO) or extracts 20fold in TBST plus 1% DMSO. Add 5 ml of the pre-dilutedcompounds/extracts to the wells described in step 6. Incubate for 20min. Controls receive no drug.

9. Start the kinase reaction by addition of 5 ml ATP mix; Shake theplates on an ELISA plate shaker during incubation.

10. Stop the kinase reaction after 60 min by addition of 30 mL stopsolution to each well.

11. Place the phosphocellulose mat and the ELISA plate in the Tomtecplate harvester. Harvest and wash the filter with the filter washsolution according to the manufacturer's recommendation. Dry the filtermats. Seal the filter mats and place them in the holder. Insert theholder into radioactive detection apparatus and quantify the radioactivephosphorous on the filter mats.

Alternatively, 40 mL aliquots from individual wells of the assay platecan be transferred to the corresponding positions on thephosphocellulose filter mat. After air drying the filters, put thefilters in a tray. Gently rock the tray, changing the wash solution at15 min intervals for 1 hour. Air-dry the filter mats. Seal the filtermats and place them in a holder suitable for measuring the radioactivephosphorous in the samples. Insert the holder into a detection deviceand quantify the radioactive phosphorous on the filter mats.

CDK2/Cyclin A—Inhibition Assay

This assay analyzes the protein kinase activity of CDK2 in exogenoussubstrate.

Reagents:

A. Buffer A: (80 mM Tris (pH 7.2), 40 mM MgCl₂), 4.84 g. Tris(F.W.=121.1 g/mol), 4.07 g. MgCl₂ (F.W.=203.31 g/mol) dissolved in 500ml H₂O. Adjust pH to 7.2 with HCl.

B. Histone H1 solution (0.45 mg/ml Histone H1 and 20 mM HEPES pH 7.2: 5mg Histone Hi (Boehinger Mannheim) in 11.111 ml 20 mM HEPES pH 7.2 (477mg HEPES (F.W.=238.3 g/mol) dissolved in 100 ml ddH₂O, stored in 1 mlaliquots at −80° C.

C. ATP solution (60 μM ATP, 300 μg/ml BSA, 3 mM DTT): 120 μl 10 mM ATP,600 μl 10 mg/ml BSA to 20 ml, stored in 1 ml liquots at −80° C.

D. CDK2 solution: cdk2/cyclin A in 10 mM HEPES pH 7.2, 25 mM NaCl, 0.5mM DTT, 10% glycerol, stored in 9 μl aliquots at −80° C.

Protocol

1. Prepare solutions of inhibitors at three times the desired finalassay concentration in ddH₂O/15% DMSO by volume.

2. Dispense 20 μl of inhibitors to wells of polypropylene 96-well plates(or 20 μl 15% DMSO for positive and negative controls).

3. Thaw Histone H1 solution (1 ml/plate), ATP solution (1 ml/plate plus1 aliquot for negative control), and CDK2 solution (9 μl/plate). KeepCDK2 on ice until use. Aliquot CDK2 solution appropriately to avoidrepeated freeze-thaw cycles.

4. Dilute 9 μl CDK2 solution into 2.1 ml Buffer A (per plate). Mix.Dispense 20 μl into each well.

5. Mix 1 ml Histone Hi solution with 1 ml ATP solution (per plate) intoa 10 ml screw cap tube. Add γ³³P ATP to a concentration of 0.15 μCi/20μl(0.15 μCi/well in assay). Mix carefully to avoid BSA frothing. Add 20 μlto appropriate wells. Mix plates on plate shaker. For negative control,mix ATP solution with an equal amount of 20 mM HEPES pH 7.2 and add γ³³pATP to a concentration of 0.15 μCi/20 μl solution. Add 20 μl toappropriate wells.

6. Let reactions proceed for 60 minutes.

7. Add 35 μl 10% TCA to each well. Mix plates on plate shaker.

8. Spot 40 μl of each sample onto P30 filter mat squares. Allow mats todry (approx. 10-20 minutes).

9. Wash filter mats 4×10 minutes with 250 ml 1% phosphoric acid (10 mlphosphoric acid per liter ddH₂O).

10. Count filter mats with beta plate reader.

Cellular/Biologic Assays

PDGF-Induced BrdU Incorporation Assay

Materials and Reagents:

(1) PDGF: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany.

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution : 1×PBS, pH 7.4 (Sugen, Inc., Redwood City,Calif.).

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line genetically engineered to express human PDGF-R.

Protocol

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (PDGF, 3.8 nM, prepared in DMEM with 0.1% BSA) andtest compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (PDGF) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

The blocking solution is removed by decanting and the wells are washedonce with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) isadded (100 μl/well) and the plate is incubated for 90 minutes at roomtemperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

EGF-Induced BrdU Incorporation Assay

Materials and Reagents

(1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan.

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1×PBS, pH 7.4 (Sugen, Inc., Redwood City,Calif.).

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line genetically engineered to express human EGF-R.

Protocol

(1) Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, ina 96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (EGF, 2 nM, prepared in DMEM with 0.1% BSA) andtest compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (EGF) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

EGF-Induced Her2-Driven BrdU Incorporation

Materials and Reagents

(1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1X PBS, pH 7.4, made in house.

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line engineered to express a chimeric receptor having theextra-cellular domain of EGF-R and the intra-cellular domain of Her2.

Protocol

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a96-well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and testcompounds are added to the cells simultaneously. The negative controlwells receive serum free DMEM with 0.1% BSA only; the positive controlcells receive the ligand (EGF) but no test compound. Test compounds areprepared in serum free DMEM with ligand in a 96 well plate, and seriallydiluted for 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

The blocking solution is removed by decanting and the wells are washedonce with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) isadded (100 μl/well) and the plate is incubated for 90 minutes at roomtemperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

IGF1-Induced BrdU Incorporation Assay

Materials and Reagents

(1) IGF1 Ligand: human, recombinant; G511, Promega Corp, USA.

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1X PBS, pH 7.4 (Sugen, Inc., Redwood City,Calif.).

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line genetically engineered to express human IGF-1receptor.

Protocol

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a96-well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (IGF1=3.3 nM, prepared in DMEM with 0.1% BSA) andtest compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (IGF1) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

(4) After 16 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

FGF-Induced BrdU Incorporation Assay

This assay measures FGF-induced DNA synthesis in 3Tc7/EGFr cells thatexpress endogenous FGF receptors.

Materials and Reagents:

1. FGF: human FGF2/bFGF (Gibco BRL, No. 13256-029).

2. BrdU Labeling reagent, (10 mM PBS (pH 7.4), Boehringer Mannheim CatNo. 1 647 229).

3. Fixdenat fixation solution (Boehringer Mannheim Cat No. 1 647 229).

4. Anti-BrdU-POD (mouse monoclonal antibody conjugated with peroxidase,Boehringer Mannheim Cat. No. 1 647 229).

5. TMB (tetramethylbenzidine, Boehringer Mannheim Cat. No. 1 647 229).

6. PBS washing solution, pH 7.4 (Sugen, Inc.).

7. Albumin, bovine (BSA), fraction V powder (Sigma Chemical Co., Cat.No. A-8551)

Procedure

1. 3T3 engineered cell line: 3T3c7/EGFr.

2. Cells are seeded at 8,000 cells/well in DMEM, 10% CS and 2 mM Gln ina 96-well plate. Incubate 24 hours at 37° C. in 5% Co₂.

3. After 24 hours, wash cells with PBS then serum starve in serum freemedium (0% DMEM, 0.1% BSA) for 24 hours.

4. Add ligand (FGF2 (1.5 nM in DMEM with 0.1% BSA) and test compoundsimultaneously. Negative control wells receive serum free DMEM with 0.1%BSA only; positive control wells receive FGF2 ligand but no testcompound. Test compounds are prepared in serum-free DMEM with ligandin.a 96-well plate and serially diluted to make seven (7) testconcentrations.

5. After 20 hours, add diluted BrdU labeling reagent (1:100 BrdU:DMEM,0.1% BSA, final concentration is 10 μM) to the cells and incubate for1.5 hours.

6. Decant medium. Remove traces of material with paper towel. AddFixDenat (50 μl/well) and incubate at room temperature for 45 minutes ona plate shaker.

7. Remove Fixdenat solution. Add blocking solution (5% dehydrated milkin PBS (200 μl/well)) and incubate for 30 minutes at room temperature ona plate shaker.

8. Decant blocking solution; wash wells once with PBS. Add anti-BrdU-PODsolution (1:100 dilution in PBS, 0.1% BSA); incubate for 90 minutes atroom temperature on a plate shaker.

9. Decant antibody conjugate; rinse wells 5 times with PBS. Dry plate byinverting on paper towel and tapping.

10. Add TMB solution (100 μl/well); incubate 20 minutes at roomtemperature on a plate shaker until color development is sufficient forphotometric detection.

11. Measure absorbance at 410 nM on a Dynatech ELISA plate reader using“Dual wavelength” mode with a filter at 490 nM.

Biochemical EGFR Assay

This assay measures the in vitro kinase activity of EGFR using ELISA.

Materials and Reagents

1. Corning 96-well Elisa plates (Corning Catalog No. 25805-96).

2. SUMO1 monoclonal anti-EGFR antibody (Biochemistry Lab, SUGEN, Inc.).

3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco Catalog No.450-1300EB).

4. TBST Buffer

Working Amount Reagent MW Concentration per L Tris 121.14  50 mM 6.057 gNaCl  58.44 150 mM 8.766 g Triton X-100 NA 0.1% 1.0 ml

5. Blocking Buffer:

Working Amount Reagent MW Concentration per 100 ml Carnation 5% 5.0 gInstant Non-Fat Milk PBS NA NA 100 ml

6. A431 cell lysate (Screening Lab, SUGEN, Inc.)

7. TBS Buffer:

Working Amount Reagent MW Concentration per L Tris 121.14  50 mM 6.057 gNaCl 58.44 150 mM 8.766 g

8. TBS + 10% DMSO

Working Amount Reagent MW Concentration per L Tris 121.14  50 mM 1.514 gNaCl  58.44 150 mM 2.192 g DMSO NA 10% 25 ml

9. Adenosine-5′-triphosphate (ATP, from Equine muscle, Sigma Cat. No.A-5394).

Prepare a 1.0 mM solution in dH₂O. This reagent should be made upimmediately prior to use and kept on ice.

10. MnCl₂.

Prepare a 1.0 M stock solution in dH₂O.

11. ATP/MnCl₂ phosphorylation mix

Stock Amount Working Reagent solution per 10 ml Concentration ATP 1.0 mM300 μl 30 μM MnCl₂ 1.0 M 500 μl 50 mM dH₂O 9.2 ml

This reagent should be prepared immediately before use and kept on ice.

12. NUNC 96-well V bottom polypropylene plates (Applied Scientific Cat.No. AS-72092).

13. Ethylenediaminetetraacetic acid (EDTA)

Prepare 200 mM working solution in dH₂O. Adjust to pH 8.0 with 10 NNaOH.

14. Rabbit polyclonal anti-phosphotyrosine serum (Biochemistry Lab,SUGEN, Inc.)

15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALIO404)

16. ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), SigmaCat. No. A-1888).

Working Amount Reagent MW Concentration per L Citric Acid 192.12 100 mM19.21 g Na2HPO4 141.96 250 mM 35.49 g ABTS NA 0.5 mg/ml 500 mg

Mix first two ingredients in about 900 ml dH₂O, adjust pH to 4.0 withphosphoric acid. Add ABTS, cover, let sit about 0.5 hr., filter. Thesolution should be kept in the dark at 4° C. until ready to use.

17. Hydrogen peroxide 30% solution (Fisher Cat. No. H325)

18. ABTS/H₂O₂

Mix 15 ml ABTS solution and 2.0 μl H₂O₂. Prepare 5 minutes before use.

19. 0.2 M HCl

Procedure

1. Coat Corning 96 well ELISA plates with 0.5 μg SUMO1 in

100 μl PBS per well, store overnight at 4° C.

2. Remove unbound SUMO1 from wells by inverting plate to remove liquid.Wash 1× with dH₂O. Pat the plate on a paper towel to remove excessliquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. atroom temperature with shaking.

4. Wash plate 3× with deionized water, then once with TBST. Pat plate ona paper towel to remove excess liquid and bubbles.

5. Dilute lysate in PBS (7 μg lysate/100 μl PBS).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 60 min.

7. Wash plates as described in 4, above.

8. Add 120 μl TBS to ELISA plate containing captured EGFR.

9. Dilute test compound 1:10 in TBS in 96-well polypropylene plates (ie.10 μl compound+90 μl TBS).

10. Add 13.5 μl diluted test compound to ELISA plate. To control wells(wells which do not receive any test compound), add 13.5 μl TBS+10%DMSO.

11. Incubate for 30 minutes while shaking at room temperature.

12. Add 15 μl phosphorylation mix directly to all wells except negativecontrol well which does not receive ATP/MnCl₂ (final well volume shouldbe approximately 150 μl with 3 μM ATP/5 mM MnCl₂ final concentration ineach well.) Incubate 5 minutes while shaking.

13. After 5 minutes, stop reaction by adding 16.5 μl of 200 mM EDTA (pH8.0) to each well, shaking continuously. After the EDTA has been added,shake for 1 min.

14. Wash 4× with deionized water, twice with TBST.

15. Add 100 μl anti-phosphotyrosine (1:3000 dilution.in TBST) per well.Incubate 30-45 min. at room temperature, with shaking.

16. Wash as described in 4, above.

17. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate(1:2000 dilution in TBST) to each well. Incubate 30 min. at roomtemperature, with shaking.

18. Wash as described in 4, above.

19. Add 100 μl of ABTS/H₂O₂ solution to each well.

20. Incubate 5 to 10 minutes with shaking. Remove any bubbles.

21. If necessary stop reaction with the addition of 100 μl 0.2 M HCl perwell.

22. Read assay on Dynatech MR7000 ELISA reader. Test Filter: 410 nMReference Filter: 630 nM

Biochemical PDGFR Assay

This assay measures the in vitro kinase activity of PDGFR using ELISA.

Materials and Reagents

Unless otherwise noted, the preparation of working solution of thefollowing reagents is the same as that for the Biochemical EGFR assay,above.

1. Corning 96-well Elisa plates (Corning Catalog No. 25805-96).

2. 28D4C10 monoclonal anti-PDGFR antibody (Biochemistry Lab, SUGEN,Inc.).

3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco Catalog No.450-1300EB)

4. TBST Buffer.

5. Blocking Buffer.

6. PDGFR-β expressing NIH 3T3 cell lysate (Screening Lab, SUGEN, Inc.).

7. TBS Buffer.

8. TBS+10% DMSO.

9. Adenosine-5′-triphosphate (ATP, from Equine muscle, Sigma Cat. No.A-5394).

10. MnCl₂.

11. Kinase buffer phosphorylation mix.

Stock Amount Working Reagent solution per 10 ml Concentration Tris 1 M250 μl 25 mM NaCl 5 M 200 μl 100 mM  MnCl₂ 1 M 100 μl 10 mM TX-100 100mM  50 μl 0.5 mM 

12. NUNC 96-well V bottom polypropylene plates (Applied Scientific Cat.No. AS-72092).

13. Ethylenediaminetetraacetic acid (EDTA).

14. Rabbit polyclonal.anti-phosphotyrosine serum (Biochemistry Lab,SUGEN, Inc.).

15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALI0404).

16. 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, SigmaCat. No. A-1888).

17. Hydrogen peroxide 30% solution (Fisher Cat. No. H325).

18. ABTS/H₂O₂.

19. 0.2 M HCl.

Procedure

1. Coat Corning 96 well ELISA plates with 0.5 μg 28D4C10 in 100 μl PBSper well, store overnight at 4° C.

2. Remove unbound 28D4C10 from wells by inverting plate to removeliquid. Wash 1× with dH₂O. Pat the plate on a paper towel to removeexcess liquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. atroom temperature with shaking.

4. Wash plate 3× with deionized water, then once with TBST. Pat plate ona paper towel to remove excess liquid and bubbles.

5. Dilute lysate in HNTG (10 μg lysate/100 μl HNTG)

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 60 min.

7. Wash plates as described in 4, above.

8. Add 80 μl working kinase buffer mix to ELISA plate containingcaptured PDGFR.

9. Dilute test compound 1:10 in TBS in 96-well polypropylene plates(i.e., 10 μl compound+90 μl TBS).

10. Add 10 μl diluted test compound to ELISA plate. To control wells(wells which do not receive any test compound), add 10 μl TBS+10% DMSO.

11. Incubate for 30 minutes while shaking at room temperature.

12. Add 10 μl ATP directly to all wells except negative control well(final well volume should be approximately 100 μl with 20 μM ATP in eachwell.) Incubate 30 minutes while shaking.

13. After 30 minutes, stop reaction by adding 10 μl of 200 mM EDTA (pH8.0) to each well.

14. Wash 4× with deionized water, twice with TBST.

15. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) per well.Incubate 30-45 min. at room temperature, with shaking.

16. Wash as described in 4, above.

17. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate(1:2000 dilution in TBST) to each well. Incubate 30 min. at roomtemperature, with shaking.

18. Wash as described in 4, above.

19. Add 100 μl of ABTS/H₂O₂ solution to each well.

20. Incubate 10 to 30 minutes with shaking. Remove any bubbles.

21. If necessary stop reaction with the addition of 100 μl 0.2 M HCl perwell.

22. Read assay on Dynatech MR7000 ELISA reader: test filter: 410 nM,reference filter: 630 nM.

Biochemical FGFR Assay

This assay measures in vitro kinase activity of the Myc-GyrB-FGFR fusionprotein using ELISA.

Materials and Reagents

1. HNTG

5x Stock Amount 1x Working Reagent MW Concentration per L ConcentrationHEPES 238.3 100 mM 23.83 g  20 mM NaCl 58.44 750 mM 43.83 g 150 mMGlycerol NA 50% 500 ml  10% Triton X-100 NA  5% 10 ml 1.0%

To make a liter of 5×stock solution, dissolve HEPES and NaCl in about350 ml dH₂O, adjust pH to 7.2 with HCl or NaOH (depending on the HEPESthat is used), add glycerol, Triton X-100 and then dH₂O to volume.

2. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco Catalog #450-1300EB)3. Blocking Buffer.

4. Kinase Buffer.

10 x Stock 1x Working Reagent MW Concentration Concentration HEPES (pH7.2) 238.3 500 mM 50 mM MnCl₂  20 mM  2 mM MgCl₂ 203.32 200 mM 10 mMTriton-X-100 1% 0.1% DTT 380.35  5 mM 0.5 mM 

5. Phenylmethylsulfonyl fluoride (PMSF, Sigma, Cat. No. P-7626):

Working solution: 100 mM in ethanol.

6. ATP (Bacterial source, Sigma Cat. No. A-7699)

Use3.31 mg per ml MilliQ H₂O for a stock concentration of 6 mM.

7. Biotin conjugated anti-phosphotyrosine mab (clone 4G10, UpstateBiotechnology Inc. Cat. No. 16-103, Ser. No. 14495).

8. Vectastain Elite ABC reagent (Avidin peroxidase conjugate, VectorLaboratories Cat. No. PK-6 100).

9. ABTS Solution.

10. Hydrogen peroxide 30% solution (Fisher Catalog #H325).

11. ABTS/H₂O₂.

12. 0.2 M HCl.

13. TRIS HCl (Fischer Cat. No. BP 152-5).

Prepare 1.0 mM solution in MilliQ H₂O, adjust pH to 7.2 with HCl.

14. NaCl (Fisher Cat. No. S271-10).

Prepare 5 M solution in MilliQ H₂O.

15. MgCl₂ (Fisher Cat. No. M33-500).

Prepare 1 M solution in MiliiQ H₂O.

16. HEPES (Fisher Cat. No. BP310-500).

Prepare 1 M solution in MilliQ H₂O, adjust pH to 7.5, sterile filter.17. TBST Buffer. 18. Sodium Carbonate Buffer (Fisher Cat. No. S495).

Prepare 0.1 M solution in MilliQ H₂O, adjust pH to 9.6 with NaOH,filter.

19. Dithiothreitol (DTT, Fisher Cat. No. BP172-25).

Prepare 0.5 mM working solution in MilliQ H₂O just prior to use. Storeat −20° C. until used, discard any leftover.

20. MnCl₂.

21. Triton X-100.

22. Goat a-Rabbit IgG (Cappel).

23. Affinity purified Rabbit a GST GyrB (Biochemistry Lab. SUGEN, Inc.).

Procedure

All of the following steps are conducted at room temperature unlessotherwise indicated.

1. Coat Corning 96-well ELISA plates with 2 μg Goat α-Rabbit antibodyper well in Carbonate Buffer such that total well volume is 100 μl.Store overnight at 4° C.

2. Remove unbound Goat a-Rabbit antibody by inverting plate to removeliquid. Pat plate on a paper towel to remove excess liquid and bubbles

3. Add 150 μl Blocking Buffer (5% Low Fat Milk in PBS) to each well.Incubate while shaking on a micro-titer plate shaker for 30 min.

4. Wash 4× with TBST. Pat plate on a paper towel to remove excess liquidand bubbles.

5. Add 0.5 μg Rabbit a-GyrB antibody per well. Dilute antibody in DPBSto a final volume of 100 μl per well. Incubate with shaking on amicro-titer plate shaker at room temperature for 1 hour.

6. Wash 4× with TBST as described in step 4.

7. Add 2 μg COS/FGFR cell lysate (Myc-GyrB-FGFR source) in HNTG to eachwell to give a final volume of 100 μl per well. Incubate with shaking ona micro-titer plate shaker for 1 hour.

8. Wash 4× with TBST as described in step 4.

9. Add 80 μl of 1×kinase buffer per well.

10. Dilute test compound 1:10 in 1× kinase buffer+1% DMSO in apolypropylene 96 well plate.

11. Transfer 10 μl of diluted test compound solution and control wellsfrom polypropylene plate wells to the corresponding ELISA plate wells,incubate with shaking on a micro-titer plate shaker for 20 minutes.

12. Add 10 μl of 70 μM ATP diluted in kinase buffer to positive controland test wells (Final ATP concentration is 7 μM/well). Add 10 μl 1×kinase buffer to negative control wells. Incubate with shaking on amicro-titer plate shaker for 15 min.

13. Stop kinase reaction by adding 5 μl 0.5 M EDTA to all wells.

14. Wash 4× with TBST as described in step 4.

15. Add 100 μl biotin conjugated a-phosphotyrosine mab (b4G10) dilutedin TBST to each well. Incubate with shaking on a micro-titer plateshaker for 30 minutes.

16. Prepare Vectastain ABC reagent. Add 1 drop reagent A to 15 ml TBST.Mix by inverting tube several times. Add 1 drop reagent B and mix again.

17. Wash 4× with TBST as described in step 4.

18. Add 100 μl ABC HRP reagent to each well. Incubate with shaking on amicro-titer plate shaker for 30 minutes.

19. Wash 4× with TBST as described in step 4.

20. Add 100 μl of ABTS/H₂O₂ solution to each well.

22. Incubate 5 to 15 minutes with shaking. Remove any bubbles.

23. If necessary stop reaction by adding 1 00 μl of 0.2M HCl/well.

24. Read assay on Dynatech MR7000 ELISA Plate Reader; test filter: 410nM, reference filter: 630 nM.

Biochemical FLK-1 Assay

This assay evaluates flk-1 autophosphorylation activity in vitro usingELISA.

Materials and Reagents

1. 15 cm tissue culture dishes

2. Flk-1/NIH cells: NIH fibroblast line over-expressing human flk-1clone 3 (SUGEN, Inc., obtained from MPI, Martinsried, Germany).

3. Growth medium: DMEM plus heat inactivated 10% FBS and 2 mM Glutamine(Gibco-BRL).

4. Starvation medium: DMEM plus 0.5% heat-inactivated FBS, 2 mMGlutamine (Gibco-BRL).

5. Corning 96-well ELISA plates (Corning Cat. No. 25805-96).

6. L4 or E38 monoclonal antibody specific for flk-1; Purified byProtein-A agarose affinity chromatography (SUGEN, Inc.).

7. PBS (Dulbecco's Phosphate-Buffered Saline) Gibco Cat. No.450-1300EB).

8. HNTG (see BIOCHEMICAL FGFR for preparation).

9. Pierce BCA protein determination kit.

10. Blocking buffer

11. TBST (pH 7.0)

12. Kinase Buffer

13. Kinase Stop Solution: 200 mM EDTA.

14. Biotinylated 4G10, specific for phosphotyrosine (UBI, Cat. No. No.16-103).

15. AB kit (Vector Laboratories Cat. No. PK 4000).

16. DMSO

17. NUNC 96-well V bottom polypropylene plates (Applied Scientific Cat.No. AS-72092).

18. Turbo-TMB (Pierce).

19. Turbo-TMB stop solution: 1 M H₂SO₄.

20. ATP (Sigma Cat. No. A-7699).

21. 20% DMSO in TBS (pH 7.0).

Procedure

Cell Growth and Lysate Preparation.

1. Seed cell into growth medium and grow for 2-3 days to 90-100%confluency at 37° C. and 5% C)₂. Do not exceed passage #20.

2. Remove the medium and wash the cells twice with PBS. Lyse with HNTGlysis buffer. Collect all lysates and vortex mix them for 20-30 seconds.

3. Remove insoluble material by centrifugation (5-10 min at about10,000×g).

4. Determine the protein concentration using BCA kit.

5. Partition lysate into 1 mg aliquots, store at −80° C.

Assay Procedure

1. Coat Corning 96-well ELISA plates with 2 μg/well purified L4 (or E38) in 100 μl of PBS. Store overnight at 4° C.

2. Remove unbound proteins from wells by inverting the plate to removethe liquid. Wash one time with dH₂O pat plate on paper towel to removeexcess liquid.

3. Block plates with 150 μl blocking buffer per well. Incubate for 45-60minutes with shaking at 4° C.

4. Remove the blocking buffer and wash the ELISA plate three times withdH₂O and one time with TBST. Pat plate on paper towel to remove excessliquid.

5. Dilute lysate in PBS to give final concentration of 50 μg/100 μl. Add100 μl of diluted lysate to each well. Incubate with shaking at 4° C.overnight.

6. Remove unbound proteins from wells by inverting the plate. Wash as instep 4.

7. Add 80 μl of kinase buffer to wells (90 μl to negative controlwells).

8. Dilute test compounds (normally 10-fold) into wells of apolypropylene plate containing 20% DMSO in TBS.

9. Add 10 μl of the diluted compounds to the ELISA wells containingimmobilized flk-1 and shake. Control wells receive no compounds.

10. From stock 1 mM ATP, prepare 0.3 mM ATP solution in dH₂₀(alternatively, kinase buffer may be used).

11. Add 10 μl of 0.3 mM ATP to all wells except the negative controls.Incubate for 60 min. at room temperature with shaking.

12. After 1 hr stop the kinase reaction by adding 11 μl 200 mM EDTA.Shake for 1-2 min.

13. Wash the ELISA plate 4 times with dH₂O and twice with TBST.

14. Add 100 μl of 1:5000 biotinylated 4G10:TBST to all wells. Incubate45 min with shaking at room temperature.

15. While the above is incubating, add 50 μl of solutions A & B from theABC kit to 10 ml of TBST. These solutions must be combined approximately30 min prior to use.

16. Wash plates as in step 4.

17. Add 100 μl of the preformed A & B complex to all wells. Incubate 30min with shaking at room temperature.

18. Wash plates as in step 4.

19. Add 100 μl turbo-TMB. Shake at room temperature for 10-15 min.

20. When the color in the positive control wells reaches an absorbanceof about 0.35-0.4, stop the reaction with 100 μl of turbo-TMB stopsolution.

21. Read plates on Dynatech MR7000 ELISA reader; test filter: 450 nM,reference filter: 410 nM.

HUV-EC-C Assay

The following protocol may also be used to measure a compound's activityagainst PDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of which arenaturally expressed by HUV-EC cells.

DAY 0

1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelialcells, (American Type Culture Collection; catalogue no. 1730 CRL). Washwith Dulbecco's phosphate-buffered saline (D-PBS; obtained from GibcoBRL; catalogue no. 14190-029) 2 times at about 1 ml/10 cm² of tissueculture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic celldissociation solution (Sigma Chemical Company; catalogue no. C-1544).The 0.05% trypsin is made by diluting 0.25% trypsin/l mM EDTA (Gibco;catalogue no. 25200-049) in the cell dissociation solution. Trypsinizewith about 1 ml/25-30 cm² of tissue culture flask for about 5 minutes at37° C. After cells have detached from the flask, add an equal volume ofassay medium and transfer to a 50 ml sterile centrifuge tube (FisherScientific; catalogue no. 05-539-6).

2. Wash the cells with about 35 ml assay medium in the 50 ml sterilecentrifuge tube by adding the assay medium, centrifuge for 10 minutes atapproximately 200×g, aspirate the supernatant, and resuspend with 35 mlD-PBS. Repeat the wash two more times with D-PBS, resuspend the cells inabout 1 ml assay medium/15 cm² of tissue culture flask. Assay mediumconsists of F12K medium (Gibco BRL; catalogue no. 21127-014) and 0.5%heat-inactivated fetal bovine serum. Count the cells with a CoulterCounter® (Coulter Electronics, Inc.) and add assay medium to the cellsto obtain a concentration of 0.8-1.0×10⁵ cells/ml.

3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8-1.0×10⁴cells/well; incubate ˜24 h at 37° C., 5% CO₂.

DAY 1

1. Make up two-fold test compound titrations in separate 96-well plates,generally 50 μM on down to 0 μM. Use the same assay medium as mentionedin day 0, step 2 above. Titrations are made by adding 90 μl/well of testcompound at 200 μM (4× the final well concentration) to the top well ofa particular plate column. Since the stock test compound is usually 20mM in DMSO, the 200 μM drug concentration contains 2% DMSO.

A diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovineserum) is used as diluent for the test compound titrations in order todilute the test compound but keep the DMSO concentration constant. Addthis diluent to the remaining wells in the column at 60 μl/well. Take 60μl from the 120 μl of 200 μM test compound dilution in the top well ofthe column and mix with the 60 μl in the second well of the column. Take60 μl from this well and mix with the 60 μl in the third well of thecolumn, and so on until two-fold titrations are completed. When thenext-to-the-last well is mixed, take 60 μl of the 120 μl in this welland discard it. Leave the last well with 60 μl of DMSO/media diluent asa non-test compound-containing control. Make 9 columns of titrated testcompound, enough for triplicate wells each for: (1) VEGF (obtained fromPepro Tech Inc., catalogue no. 100-200; (2) endothelial cell growthfactor (ECGF) (also known as acidic fibroblast growth factor, or aFGF)(obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600);or, (3) human PDGF B/B (1276-956, Boehringer Mannheim, Germany) andassay media control. ECGF comes as a preparation with sodium heparin.

2. Transfer 50 μl/well of the test compound dilutions to the 96-wellassay plates containing the 0.8-1.0×10⁴ cells/100 μl/well of theHUV-EC-C cells from day 0 and incubate ˜2 h at 37° C., 5% CO₂.

3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, ormedia control to each test compound condition. As with the testcompounds, the growth factor concentrations are 4× the desired finalconcentration. Use the assay media from day 0 step 2 to make theconcentrations of growth factors. Incubate approximately 24 hours at 37°C., 5% CO₂. Each well will have 50 μl test compound dilution, 50 μlgrowth factor or media, and 100 μl cells, which calculates to 200μl/well total. Thus the 4× concentrations of test compound and growthfactors become 1× once everything has been added to the wells.

DAY 2

1. Add ³H-thymidine (Amersham; catalogue no. TRK-686) at 1 μCi/well (10μl/well of 100 μCi/ml solution made up in RPMI media+10%heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5%CO₂. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.

DAY 3

1. Freeze plates overnight at −20° C.

DAY 4

Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester96®) onto filter mats (Wallac; catalogue no. 1205-401); read counts on aWallac Betaplate liquid scintillation counter.

In Vivo Animal Models

Xenograft Animal Models

The ability of human tumors to grow as xenografts in athymic mice (e.g.,Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xeno-transplantation of human tumors into athymic mice,(Rygaard and Poylsen, 1969, Acta Pathol. Microbial. Scand., 77:758-760),many different human tumor cell lines (e.g., mammary, lung,genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a TK in a cell. The object of the in vivo assaysis to determine the effect of a compound in an animal model of aparticular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC #CCL 107), A375 cells (melanoma, ATCC #CRL 1619),A431 cells (epidermoid carcinoma, ATCC #CRL 1555), Calu 6 cells (lung,ATCC #HTB 56), PC3 cells (prostate, ATCC #CRL 1435), SKOV3TP5 cells andNIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR,IGF-1R or any other test kinase. The following protocol can be used toperform xenograft experiments:

Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water ad libitum.

Cell lines are grown in appropriate medium (for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90-95% air and 5-10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for 10 min. Pellets are resuspended in sterile PBS ormedia (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8-10 mice per group, 2-10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product oflength×width×height unless otherwise indicated. P values are calculatedusing the Students t-test. Test compounds in 50-100 μL excipient (DMSO,or VPD:D5W (U.S. Pat. No. 5,610,173 to Sxhwartz, et al.), can bedelivered by IP injection at different concentrations generally startingat day one after implantation.

Tumor Invasion Model

The following tumor invasion model has been developed and may be usedfor the evaluation of therapeutic value and efficacy. of the compoundsidentified to selectively inhibit KDR/FLK-1 receptor.

Procedure

8 week old nude mice (female) (Simonsen Inc.) are used as experimentalanimals. Implantation of tumor cells can be performed in a laminar flowhood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and5 mg/kg Xylazine) are administered intraperitoneally. A midline incisionis done to expose the abdominal cavity (approximately 1.5 cm in length)to inject 10⁷ tumor cells in a volume of 100 μl medium. The cells areinjected either into the duodenal lobe of the pancreas or under theserosa of the colon. The peritoneum and muscles are closed with a 6-0silk continuous suture and the skin is closed by using wound clips.Animals are observed daily.

Analysis

After 2-6 weeks, depending on gross observations of the animals, themice are sacrificed, and the local tumor metastases to various organs(lung, liver, brain, stomach, spleen, heart, muscle) are excised andanalyzed (measurement of tumor size, grade of invasion, immunochemistry,in situ hybridization determination, etc.).

Measurement of Cell Toxicity

Therapeutic compounds should be more potent in inhibiting receptortyrosine kinase activity than in exerting a cytotoxic effect. A measureof the effectiveness and cell toxicity of a compound can be obtained bydetermining the therapeutic index; i.e., IC₅₀/LD₅₀. IC₅₀, the doserequired to achieve 50% inhibition, can be measured using standardtechniques such as those described herein. LD₅₀, the dosage whichresults in 50% toxicity, can also be measured by standard techniques aswell (Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring theamount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol.Methods, 64:313; Decker and Lohmann-Matthes, 1988, J. Immunol. Methods,115:61), or by measuring the lethal dose in animal models. Compoundswith a large therapeutic index are preferred. The therapeutic indexshould be greater than 2, preferably at least 10, more preferably atleast 50.

CONCLUSION

It will be appreciated that the compounds, methods and pharmaceuticalcompositions of the present invention are effective in modulating PKactivity and therefore are expected to be effective as therapeuticagents against RTK, CTK-, and STK-related disorders.

One skilled in the art would also readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent herein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein.are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional. features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

Other embodiments may be found within the following claims.

What is claimed:
 1. A 2-indolinone having the chemical structure I, II,or III:

or a physiologically acceptable salt of the compound, wherein: R¹ isselected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, hydroxy, alkoxy, —C(═O)OR″, R″C(═O)O—, C-amido,C-thioamido, acetyl, —S(═O)₂R″, and trihalomethylsulfonyl, wherein R″ isselected from the group consisting of hydrogen alkyl, cycloalkyl, aryl,heteroaryl (bonded through a ring carbon), and heteroalicyclic (bondedthrough a ring carbon); A, B, D and E are independently selected fromthe group consisting of carbon and nitrogen, with the proviso that atleast one of A, B, D, or E is nitrogen; wherein when A, B, D or E isnitrogen, R², R³, R⁴ or R⁵, respectively does not exist; F, G, J and Kare independently selected from the group consisting of carbon,nitrogen, oxygen and sulfur, wherein when n is 1 and F, G, J or K is anatom other than carbon, R⁶, R⁷, R⁸ or R⁹, respectively, does not exist;when n is 0 and F, G or K is oxygen or sulfur, R⁶, R⁸ or R⁹,respectively, does not exist; R², R³, R⁴, R⁵ R⁶, R⁷, R⁸ and R⁹ areindependently selected from the group consisting of hydrogen, alkyl,trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,arylthio, —S(═O)R″, —S(═O)₂R″, S-sulfonamido, N-sulfonamido,N-trihalomethanesulfonamido, —C(═O)R″, —C(═O)OR″, R″C(═O)O—, cyano,nitro, halo, cyanato, isocyanato, thiocyanato, isothiocyanato,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, amino and —NR¹³R¹⁴; R² and R³ or R³ and R⁴ or R⁴ and R⁵ or R⁶and R⁷ or R⁷ and R⁸ or R⁸ and R⁹ may combine to form a methylenedioxy oran ethylenedioxy group; R¹³ and R¹⁴ are independently selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, —C(═O)R″, acetyl, —S(O)₂R″, trihalomethanesulfonyl and,combined, a five-member or a six-member heteroalicyclic ring; R¹⁰, R¹¹and R¹² are independently selected from the group consisting of alkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, cyano,trihalomethyl, hydroxy, alkoxy, alkylthio, aryloxy, arylthio, R″C(═O)O—,—C(═O)OR″, —C(═O)O⁻M⁺, —(CH₂)_(r)C(═O)OR″, —(CH₂)_(r)C(═O)O⁻M⁺, C-amido,N-amido, cyanato, isocyanato, thiocyanato, isothiocyanato, amino,—S(═O)R″, —S(═O)₂R″, nitro and —NR¹³R¹⁴; R¹⁰ and R¹¹ or R¹² and R¹² maycombine to form an endo double bond; Z is selected from the groupconsisting of oxygen and sulfur; r is 1, 2, 3, 4, 5 or 6; and, n is 0 or1; it being understood that when n is 0, there are no more than 2 doublebonds within the ring comprising F, G and K.
 2. The compound or salt ofclaim 1, wherein F, G, J and K are carbon.
 3. The compound or salt ofclaim 1, wherein: F is nitrogen; R⁹ is hydrogen; and, G and K arecarbon.
 4. The compound or salt of claim 1, wherein; K is nitrogen; R⁶is hydrogen; and, F and G are carbon.
 5. The compound or salt of claim1, wherein one or two of F, G, J or K are independently nitrogen.
 6. Thecompound or salt of claim 1, wherein: R¹³ is hydrogen; and, R¹⁴ isunsubstituted lower alkyl.
 7. The compound or salt of claim 1, wherein:R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selected from thegroup consisting of: hydrogen; unsubstituted lower alkyl; lower alkylsubstituted with a group selected from the group consisting of halo,—C(═O)OR″ and —NR¹³R¹⁴; unsubstituted lower alkoxy; lower alkoxysubstituted with a group selected from the group consisting of halo,—C(═O)OR″, unsubstituted aryl or —NR¹³R¹⁴; trihalomethyl; unsubstitutedalkenyl; unsubstituted alkynyl; unsubstituted aryl; aryl substitutedwith one or more groups independently selected from the group consistingof unsubstituted lower alkyl or lower alkyl substituted with a groupselected from the group consisting of halo, —C(═O)OR″ or NR¹³R¹⁴;unsubstituted heteroalicyclic; heteroalicyclic substituted with one ormore groups independently selected from the group consisting ofunsubstituted lower alkyl, —C(═O)H, —C(═O)—(unsubstituted lower alkyl),hydroxy, unsubstituted alkoxy, alkoxy substituted with a group selectedfrom the group consisting of halo, —C(═O)OR″ and —NR¹³R¹⁴; unsubstitutedaryloxy; aryloxy substituted with a group independently selected fromthe group consisting of unsubstituted lower alkyl, trihalomethyl, halo,hydroxy and amino; mercapto; unsubstituted alkylthio; unsubstitutedarylthio; arylthio substituted with one or more groups independentlyselected from the group consisting of halo, hydroxy and amino;S-sulfonamido; —C)═O)OR″; R″C(═O)O—; hydroxy; cyano; nitro; halo;C-amido; N-amido; amino; and, —NR¹³R¹⁴.
 8. The compound or salt of claim6 wherein: R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selectedfrom the group consisting of: hydrogen; unsubstituted lower alkyl; loweralkyl substituted with a group selected from the group consisting ofhalo, —C(═O)OR″ and —NR¹³R¹⁴; unsubstituted lower alkoxy; lower alkoxysubstituted with a group selected from the group consisting of halo,—C(═O)OR″, unsubstituted aryl or —NR¹³R¹⁴; trihalomethyl; unsubstitutedalkenyl; unsubstituted alkynyl; unsubstituted aryl; aryl substitutedwith one or more groups independently selected from the groupsconsisting of unsubstituted lower alkyl or lower alkyl substituted witha group selected from the group consisting of halo, —C(═O)OR″ or—NR¹³R¹⁴; unsubstituted heteroalicyclic; heteroalicyclic substitutedwith one or more groups independently selected from the group consistingof unsubstituted lower alkyl, —C(═O)H, —C(═O)—(unsubstituted loweralkyl), hydroxy, unsubstituted alkoxy, alkoxy substituted with a groupselected from the group consisting of halo, —C(═O)OR″ and —NR¹³R¹⁴;unsubstituted aryloxy; aryloxy substituted with one or more groupsindependently selected from the group consisting of unsubstituted loweralkyl, trihalomethyl, halo, hydroxy and amino; mercapto; unsubstitutedalkylthio; unsubstituted arylthio; arylthio substituted with one or moregroups independently selected from the group consisting of halo, hydroxyand amino; S-sulfonamido; —C(═O) O^(R″;) R″C(═O)O—; hydroxy; cyano;nitro; halo; C-amido; N-amido; amino; and, —NR¹³R¹⁴.
 9. The compound orsalt of claim 1, wherein: R¹⁰, R¹¹ and R¹² are independently selectedfrom the group consisting of hydrogen, unsubstituted lower alkyl,—(CH₂)_(r)C(═O)OR″, (CH₂)_(r)C(═O)O⁻M⁺, halo, hydroxy, alkoxy,R″C(═O)O—, —C(═O))OR″, —C(═O)O⁻M⁺, amino, C-amido, N-amido, nitro and—NR¹³R¹⁴.
 10. The compound or salt of claim 6, wherein: R¹⁰, R¹¹ and R¹²are independently selected from the group consisting of hydrogen,unsubstituted lower alkyl, —(CH₂)_(r)C(═O)OR″, —(CH₂)_(r)C(═O)O⁻M⁺,halo, hydroxy, alkoxy, R″C(═O)O—, —C(═O)OR″, —C(═O)O⁻M⁺, amino, C-amido,N-amido, nitro and —NR¹³R¹⁴.
 11. The compound or salt of claim 10wherein at least one of R¹⁰, R¹¹ or R¹² is selected from the groupconsisting of —C(═O)OR″, —C(═O)O⁻M⁺, —(CH₂)_(r)C(═O)OR″ and—(CH₂)_(r)C(═O)O⁻M⁺.
 12. The compound or salt of claim 11 wherein r ofthe —(CH₂)_(r)C(═O)OR″ or —(CH₂)_(r)C(═O)O⁻M⁺ group is
 1. 13. Thecompound or salt of claim 11 wherein r of the —(CH₂)_(r)C(═O)OR″ or(CH₂)_(r)C(═O)O⁻M⁺ group is
 2. 14. The compound or salt of claim 1,wherein said S-sulfonamido group is N,N-dimethylsulfonamido.
 15. Apharmaceutical composition, comprising: a therapeutically effectiveamount of the compound or salt of claim 1; and a pharmaceuticallyacceptable carrier or excipient.
 16. A method for the modulation of thecatalytic activity of a protein kinase comprising contacting saidprotein kinase with a compound or salt of claim
 1. 17. The method ofclaim 16 wherein said protein kinase is selected from the groupconsisting of a receptor tyrosine kinase, a non-receptor tyrosine kinaseand a serine-threonine kinase.
 18. A method for treating a proteinkinase related disorder in an organism comprising administering atherapeutically effective amount of a compound or salt of claim 1 tosaid organism.
 19. The method of claim 18 wherein said protein kinaserelated disorder is selected from the group consisting of a receptortyrosine kinase related disorder, a non-receptor tyrosine kinase relateddisorder and a serine-threonine kinase related disorder.
 20. The methodof claim 18 wherein said protein kinase related disorder is selectedfrom the group consisting of an EGFR related disorder, a PDGFR relateddisorder, an IGFR related disorder and a flk/KDR related disorder. 21.The method of claim 18 wherein said protein kinase related disorder is acancer selected from the group consisting of squamous cell carcinoma,astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladdercancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer,breast cancer, small-cell lung cancer, glioma, colorectal cancer,genitourinary cancer and gastrointestinal cancer.
 22. The method ofclaim 18 wherein said protein kinase related disorder is selected fromthe group consisting of diabetes, an autoimmune disorder, ahyperproliferation disorder, restenosis, fibrosis, psoriasis, vonHippel-Lindau disease, osteoarthritis, rheumatoid arthritis,angiogenesis, an inflammatory disorder, an immunological disorder and acardiovascular disorder.
 23. The method of claim 18 wherein saidorganism is a human.